histology and histopathology

Transcription

HISTOLOGY AND
HISTOPATHOLOGY
Cellular and Molecular Biology
Volume 26 (supplement 1), 2011
HISTOLOGY AND HISTOPATHOLOGY
http://www.hh.um.es
This Journal publishes works in all fields of microscopical
morphology; high quality is the overall consideration.
HISTOLOGY AND HISTOPATHOLOGY is an international
journal, the purpose of which is to publish original works in
English in histology, histopathology and cell biology. Its format
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TERMIS EU 2011 Annual Meeting
Tissue Engineering & Regenerative Medicine International Society
TERMIS-EU 2011 Meeting. Granada, Spain
V EUROPEAN CHAPTER
OF THE TISSUE ENGINEERING
AND REGENERATIVE MEDICINE INTERNATIONAL SOCIETY
(TERMIS)
IN CONJUNCTION
WITH XVI MEETINGOF THE SPANISH SOCIETY
OF HISTOLOGY AND TISSUE ENGINEERING
GRANADA, SPAIN, JUNE 7TH – 10TH 2011
I
II
HONORARY COMMITTEE
His Royal Highness Felipe de Borbón y Grecia, Prince of Asturias
Her Royal Highness Letizia Ortiz Rocasolano, Princess of Asturias
Cristina Garmendia Mendizábal, Minister of Science and Innovation
Leire Pajín Iraola, Minister of Health and Social Policy
José Antonio Griñán Martínez, President of the Regional Government of Andalusia
María Jesús Montero Cuadrado, Andalusian Regional Minister of Health
José Torres Hurtado, Mayor of the City of Granada
Manuel Díaz-Rubio, President of the Royal National Academy of Medicine of Spain
José Jerónimo Navas Palacios, Director of the National Institute of Health Carlos III
Francisco González Lodeiro, Rector of the University of Granada
Indalecio Sánchez-Montesinos García, Dean of the Medical School, University of Granada
María del Carmen Maroto Vela, President of the Royal Academy of Medicine of Oriental Andalusia
Natividad Cuende Melero, Executive Director of the Andalusian Inititative for Advanced Therapies
ORGANIZING COMMITTEE
Antonio Campos, Chairman
Miguel Alaminos, Vice-Chairman
Ana Celeste Ximénes Oliveira
Antonio Fernández-Montoya
Camilo Alfonso
Carlos Martínez Gómez
Giuseppe Scionti
Ingrid Johanna Garzón
José Manuel García
María del Carmen Sánchez-Quevedo
María Dolores Caracuel
Miguel Ángel Martín Piedra
Miguel González-Andrades
Olga Roda
Pascual Vicente Crespo
Renato Nieto Aguilar
Ricardo Fernández Valadés
Salvador Arias Santiago
Salvador Oyonarte
Víctor Carriel
III
INTERNATIONAL SCIENTIFIC COMMITTEE
Rui L. Reis, President
Abhay Pandit
Aldo R Boccaccini
Alexandra P. Marques
Jöns Hilborn
José Manuel García-Aznar
José Peña-Amaro
Alice Warley
Juan F. Madrid
Alicia El Haj
Julia Buján
Álvaro Meana
Karl-Heinz Schuckert
Andres Castell
Kohji Nishida
Antonios Mikos
Manuel Toledano
Carmen Carda
Manuela Gomes
Chris Mason
Claudio Migliaresi
David Williams
Erhan Piskin
María del Mar Pérez
Mauro Alini
Michael Fehlings
Michael Raghunath
Eugenio Velasco
Nuno Neves
Eva Sykova
Paul Hatton
Frank Emmrich
Ranieri Cancedda
Gerardo Catapano
Raquel Osorio
Gerjo van Osch
Robert Brown
Gilson Khang
Heinz Redl
Ismael Rodríguez
Ivan Martin
João F. Mano
Sebastián Sanmartín
Telma Zorn
Teruo Okano
Ulrich Nöth
Yves Bayon
IV
TERMIS-EU Continental Council
Continental Chair
Rui Reis
Continental Chair Elect
Alicia El-Haj
Member-At-Large
C. James Kirkpatrick
Continental Council Members
Mauro Alini
Catarina Alves
Andrea Banfi
Ranieri Cancedda
Antonio Campos
Eric Farrell
Paul Hatton
Claudio Migliaresi
Bojana Obradovic
Abhay Pandit
Erhan Piskin
Heinz Redl
Rogerio Pirraco
V EUROPEAN CHAPTER OF THE TISSUE ENGINEERING
AND REGENERATIVE MEDICINE INTERNATIONAL SOCIETY
(TERMIS)
GRANADA, SPAIN, JUNE 7TH – 10TH 2011
ABSTRACTS
A. SESSIONS AND SYMPOSIA INDEX:
1
ADIPOSE TISSUE DERIVED STEM CELLS IN TISSUE ENGINEERING
APPROACHES
2
BIOFABRICATION FOR REGENERATIVE MEDICINE APPLICATIONS
3
BIOFUNCTIONAL MATERIALS AS EXTRACELLULAR SIGNALS TO PROMOTE
TISSUE MORPHOGENESIS
4
BIOINTERFACIAL ENGINEERING IN REGENERATIVE MEDICINE
5
BIOMATERIALS & ENGINEERED CONSTRUCTS-OUTCOMES IN MEDICINE/
EXISTENT SURGERY (BECOMES)
6
BIOMATERIALS AND THE REACTIONS THEY ELICT IN THE BODY
7
BIOREACTORS TECHNOLOGIES FOR TISSUE ENGINEERING
8
CARTILAGE
9
CELL TRACTION: THE PROS AND CONS IN VALVULAR AND
VASCULAR TISSUE ENGINEERING
10
CELL VIABILITY AND TISSUE BANKING
11
CELL-BASED THERAPIES AT BED-SIDE
12
CHARACTERIZATION OF TISSUE MECHANICS
13
COMMERCIALIZING CELL THERAPIES. TRAGEDY, TUMULT AND TRIUMPH
14
COMPUTATIONAL MODELING IN TISSUE ENGINEERING
15
ENGINEERED HYDROGELS (AND STEM CELLS) FOR TISSUE
REGENERATION
16
ENGINEERING A FUNCTIONAL TENDON
17
ENGINEERING BIOMIMETIC SCAFFOLDS FOR IN VITRO STUDIES AND
REGENERATIVE THERAPIES
18
ESB - TERMIS SYMPOSIUM: BIOMECHANICS IN TISSUE ENGINEERING
19
EUROSTEC: PROGRESS AND FUTURE ASPECTS OF SOFT TISSUE
ENGINEERING FOR CHILDREN
20
EXTRACELLULAR MATRIX: FROM DEVELOPMENT BIOLOGY AT TISSUE
ENGINEERING
21
INJECTABLE SCAFFOLDS
22
INNOVATIONS IN STEM CELL-BASED CARDIAC TISSUE ENGINEERING
23
KOREAN-EUROPEAN SYMPOSIUM: BIOACTIVE SCAFFOLDS FOR TISSUE
REGENERATION
24
KOREAN-EUROPEAN SYMPOSIUM: STEM-CELL BASED TISSUE
ENGINEERING
V
25
MANUFACTURING AND CHARACTERIZATION OF SCAFFOLDS,
BASED ON POLYLACTIC ACID FIBRILS
26
MASTERING SURFACE ASPECTS TO CONTROL BIOMATERIALS
INTERACTIONS WITH CELLS AND TISSUES
27
MECHANICAL BEHAVIOUR OF CELLS, SCAFFOLDS, AND ENGINEERED
TISSUES
28
MICROVASCULAR ENGINEERING
29
MODULATING IN VITRO MICROENVIRONMENTS TO LET CELLS THRIVE:
FROM PATHOLOGY TO PHYSIOLOGY AND THERAPY
30
NANOSTRUCTURED & BIOMIMETIC SCAFFOLDS FOR SKELETAL TISSUE
ENGINEERING
31
NANOTECHNOLOGY AND REGENERATIVE MEDICINE
32
NEURAL TISSUE REGENERATION
33
PHYSICAL METHODS AND TECHNIQUES FOR THE EVALUATION AND
QUALITY CONTROL OF BIOMATERIALS AND ARTIFICIAL TISSUES
34
PLACENTAL TISSUES - A NEW AVENUE IN REGENERATIVE MEDICINE
35
POLYMERIC VECTORS FOR GENE THERAPY
36
RECENT DEVELOPMENTS IN SCAFFOLDING TECHNOLOGIES AND CELL
BASED THERAPIES IN SPINAL CORD INJURY REGENERATION
37
RELATING IN VIVO BIOCOMPATIBILITY WITH IN VIVO OUTCOME
38
RELEVANT MODELS FOR PRE-CLINICAL EVALUATION ON THE PATH TO
CLINICAL TRANSLATION
39
REPAIR, REPLACE AND REGENERATION IN THE EYE
40
CANCER AND TISSUE ENGINEERING
41
STEM CELL AND TISSUE ENGINEERING THERAPIES TO ACCOMPLISH
REGENERATIVE DENTISTRY
42
THE EXTRACELLULAR MATRIX IN TISSUE ENGINEERING: PASSIVE OR
ACTIVE PLAYER?
43
THE SPANISH CELL THERAPY NETWORK ACTIVITIES: FROM BENCH TO
BEDSIDE
44
THE USE OF MAGNETIC NANOPARTICLES FOR TAGGING, TRACKING AND
ACTIVATION IN REGENERATIVE MEDICINE
45
TISSUE ENGINEERING IN UROLOGY
46
TISSUE ENGINEERING OF SKIN: FROM BASIC RESEARCH TO
NOVEL THERAPIES
47
TRANSLATIONAL BONE ENGINEERING
48
MESENCHYMAL STEM CELLS (MSC)
49
GENERAL SESSION
50
SPANISH SOCIETY OF HISTOLOGY AND TISSUE ENGINEERING (SEHIT)
SATELLITE MEETING
B. INDUSTRY DAY: CELLS AND TISSUES AS THERAPEUTIC TOOLS
VI
1. ADIPOSE TISSUE DERIVED STEM
CELLS IN TISSUE ENGINEERING
APPROACHES
Chair: Manuela E. Gomes
Co-chair: Rui L. Reis
Keynote speaker: Jeffrey Gimble
Organizer: Manuela E. Gomes
Synopsis: In 2001 it was reported for the first time the
existence of stem cells within the adipose tissue, and
since then, this tissue has been gaining an increased
importance as a stem cells source for a wide range of
potential applications in Tissue Engineering and
Regenerative Medicine. Adipose tissue is probably the
most abundant and accessible source of adult stem cells
and thus it holds great promise for use in tissue repair
and regeneration. In fact, Adipose Stem Cells (ASCs),
present several advantages over other adult stem cell
sources, such as the bone marrow, as they can be
obtained in larger quantities, under local anesthesia and
with minimal discomfort. Furthermore, it has been
demonstrated that adipose tissue-derived stem cells
(ASCs) possess multiple differentiation capacities.
Nevertheless, to take full advantage of this cell source for
Tissue Engineering applications, current research has
been addressing several issues, such as, for example, the
differences found in the harvesting methods, differences
in fat tissue derived from different anatomic sites and the
heterogeneity of the cells population that are obtained
using the isolation methods most commonly used do far.
Many researcher have focused essentially in their
potential use in a number of regenerative medicine
approaches, based o their wide availability, possibility of
autologous use and differentiation potential.
In summary, the aim of this Symposium is to expose most
recent findings and knowledge generated from research
on adipose derived stem cells, focused on their
application in tissue engineering/regeneration.
(1.KP) CURRENT OPPORTUNITIES AND CHALLENGES IN
ADVANCING HUMAN ADIPOSE-DERIVED CELLS TO THE
CLINIC
Gimble JM (1)
1. Stem Cell Biology Laboratory, Pennington Biomedical
Research Center, Louisiana State University System, Baton
Rouge, LA 70808, USA
Subcutaneous fat has emerged as an alternative tissue
source for stromal/stem cells in regenerative medicine.
Over the past decade, international research efforts have
established a wealth of basic science and pre-clinical
evidence regarding the differentiation potential and
regenerative properties of both freshly processed,
heterogeneous stromal vascular fraction (SVF) cells and
culture expanded, relatively homogeneous adiposederived stromal/stem cells (ASCs). The SVF cells and ASC
populations display distinct advantages and functional
properties making them attractive for either autologous
or allogeneic use. Mechanistically, the cells can act via
direct differentiation to the tissue of interest and/or as a
source of trophic factors. The stage has been set for
clinicians to translate adipose-derived cells from the
I
bench to the bedside; however, this process will involve
“development” steps that fall outside of traditional
“hypothesis-driven, mechanism-based” paradigm. It is
important for the tissue engineering community to design
and pursue randomized and controlled clinical trials with
long term follow up. An evidence-based medical
approach will advance the field more effectively than
anecdotal or uncontrolled reports. Clinical applications
will be further served by standardization and
reproducibility of adipose-derived cell therapies with
respect to their efficacy and safety.
(1.O1) TNF-TREATED ADIPOSE TISSUE-DERIVED STEM
CELLS INCREASE THE MIGRATORY ACTIVITY OF
ENDOTHELIAL CELLS IN VITRO
Salamon A (1), Ramer R (2), Adam S (1), Rychly J (3),
Peters K (1)
1. Junior Research Group, Department of Cell Biology,
Medical Faculty, University of Rostock; 2. Institute of
Toxicology and Pharmacology, Medical Faculty, University
of Rostock; 3.Department of Cell Biology, Medical Faculty,
University of Rostock
Introduction. Adipose tissue-derived stem cells (ASC)
express the mesenchymal stem cell (MSC) markers CD44,
CD68, CD105 and CD166 and can differentiate along
different lineages. Since MSC are known to have
immunomodulatory effects and since freshly isolated ASC
express the perivascular marker CD34, we investigated
whether inflammatory stimulation of ASC influences
migration of human dermal microvascular endothelial
cells (HDMEC).
Materials and Methods. To this end, we treated ASC with
tumor necrosis factor (TNF), transferred the cell culture
supernatant to a culture of HDMEC and observed the
migratory activity of the endothelial cells in Scratch and
Boyden Chamber Assays. ELISA-based techniques were
used to find factors that are secreted by ASC.
Results. We found that ASC-conditioned medium
significantly increased the migratory activity of HDMEC
both in Scratch and Boyden Chamber Assays. Under TNF
treatment, ASC-mediated migratory activation of HDMEC
was further increased. Out of 31 factors that were
analyzed by ELISA-based techniques, ASC were found to
secrete 18 to the supernatant, and 13 of those factors
were more strongly secreted following TNF treatment.
Conclusion. Our findings indicate that there is an indirect
interaction between ASC and HDMEC via diverse soluble
factors. Although we can so far not decipher the
individual contributions of the large variety of factors
involved, we can nevertheless assume that ASC in vivo
modify HDMEC-mediated processes such as e. g. wound
healing, tissue infiltration by leukocytes or the
development of new blood vessels. Therefore, ASC are a
promising source for cell-based regenerative therapies.
This work was financially supported by the Ministry of
Economy, Labor and Tourism Mecklenburg-Vorpommern
and by the European Union (ESF/IV-WM-B34-0006/08).
Keywords. ASC, HDMEC, inflammation, migration
Pedro P. Carvalho acknowledges the Portuguese
Foundation for Science and Technology (F.C.T.) for his
grant (SFRH/BD/44128/2008).
Keywords. Adipose-derived stem cells; lipoaspirates;
animal-free; trypsin
Day 0
Figure 1: Conditioned medium from TNF-treated ASC
significantly increased HDMEC migratory activity.
Migration of HDMEC through 8 µm pores and towards
ASC-conditioned medium was assessed in a classical
Boyden chamber assay.
(1.O2) DEVELOPING OPTIMIZED METHODS FOR CGMP
COMPLIANCE IN THE ISOLATION OF HUMAN ADIPOSEDERIVED STROMAL/STEM CELLS
Carvalho PP (1), Yu G (2), Wu X (2), Dias IR (3), Gomes ME
(1), Reis RL (1), Gimble JM (2)
1. 3B's Research Group; 2. Pennington Biomedical
Research Center; 3. University of Tras-os-Montes e Alto
Douro
Introduction. This study aimed to explore non-animal
sources of trypsin-like enzymes as alternatives to porcine
trypsin for the passage of ASCs and to determine the
effect of time delays on the yield and function of ASCs
after collagenase digestion.
Materials and Methods. Differentiation ASCs (P1) were
induced with either Adipogenic Medium or Osteogenic
Medium for 9-12 days and stained with Oil Red O or
Alizarin Red (respectively). Flow Cytometry: hASCs were
assessed with CD29, CD34, CD44, CD45, CD73, CD90,
CD105 and IgG1 control.
Results. Trypsin alternatives. There is no significant
difference between Trypsin and animal-free alternatives
tested, in total cell recovered number and their viability;
immunophenotype and differentiation capacity in
adipogenic and osteogenic lineages is maintained.
Lipoaspirate storage, show significant differences
between total number of nucleated cells obtained in SVF
harvested on day 0 relative to days 1, 2 and 3 (room
temperature). There was no significant difference
between ASC yields on day 0 and day 1. Flow cytometric
analysis showed no significant difference in the
immunophenotype of ASCs throughout the four day
period. Capacity for adipogenic and osteogenic
differentiation remained present in cells harvested up to
day 3 although a decrease in the intensity of the staining
was evident in days 2 and 3.
Conclusions. We conclude that TrypLE Express and
TrypZean can be used in cell culture protocols as effective
animal-free alternatives to Trypsin/EDTA. Cell yield,
viability and phenotype will remain the same as cells
treated with Trypsin/EDTA. Our findings indicate that one
can obtain hASCs even 72hrs after surgical procedure but
the cell yield and differentiation ability is optimal within
the first 24hrs. These studies have relevance to the
optimization of GMP methods using ASCs in tissue
engineering and regenerative medicine.
Day 1
Day 2
Day 3
CD29 88.0 ± 20.9 96.5 ± 2.0 92.7 ± 6.6 89.9 ± 13.
CD34 91.6 ± 3.4
91.7 ± 5.2 84.9 ± 6.9 84.1 ± 5.9
CD44 11.8 ± 4.9
20.3 ± 4.4 15.1 ± 11.
8.5 ± 5.6
CD45 17.3 ± 7.4
17.5 ± 3.9 8.4 ± 4.2 * 6.0 ± 3.3 *
CD73 79.3 ± 9.2
76.1 ± 8.0 67.5 ± 21. 75.1 ± 19.
CD90 86.9 ± 5.5
80.9 ± 7.7 74.3 ± 26. 68.8 ± 27.
CD105 94.2 ± 5.5
93.9 ± 3.3 94.8 ± 6.2 96.7 ± 4.1
Table 1 – Flow Cytometry data. The percentage of
positive cells for each marker is presented as an average
value ± SD in a total of four different donors (n=4). Values
with significant difference to day 0 are marked with an
asterisk* (p<0.05).
(1.O3) KERATIN BIOMATERIALS SUPRESS PPARΓ
EXPRESSION AND ENHANCE HUMAN ADIPOSE DERIVED
STEM CELL OSTEOGENESIS
Teli T (1), Van Dyke ME (2)
1. Department of Orthopaedic Surgery, Wake Forest
Health Sciences, Winston-Salem, NC, USA; 2.Wake Forest
Institute for Regenerative Medicine, Winston-Salem, NC,
USA
Introduction. The combination of autologous,
multipotent mesenchymal stem cells (MSC) and a
biomaterial carrier has been proposed as a treatment for
patients with fractures, osteoporosis, and cancer.
However, the target patient population is typically
elderly, and significant changes in the multipotency of
MSC with age have brought the clinical utility of this
treatment paradigm into question. Specifically,
peroxisome proliferator-activated receptor gamma 2
(PPARγ2), has been shown to activate adipogenic and
suppress osteogenic differentiation pathways in aged
mice, thereby limiting the potential effectiveness of
regenerative treatments that rely on endogenous and
exogenous MSC for bone repair. The goal of this study
was to examine the effect of keratin biomaterials on
osteogenesis and PPARγ signaling in human adipose
derived stem cells (hADSC), a clinically important source
of autologous MSC for therapy.
Materials and Methods. Keratin was extracted from
human hair obtained from a commercial supplier under
oxidative conditions to yield a crude keratose mixture.
Fractions of alpha and gamma keratose were purified and
added as supplements at 0.03 mg/mL in bone
differentiation media (100nM dexamethasone, 50mM
ascorbic acid and 10mM β- glycerophosphate). hADSC
were isolated from adipose tissue collected from donors
undergoing elective abdominoplasty, assayed for CD13,
CD44, CD73, CD105 and CD166, and subsequently
induced to differentiate towards osteogenic lineage for
21 days. Cultures were assayed for alkaline phosphatase
(AP) activity and deposition of calcified mineral.
Quantitative RT-PCR assays were performed using
osteogenic-specific primers runt-related transcription
factor 2 (Runx2), collagen type I alpha 1 (Col1a1), AP, OC,
homeobox protein DLX5 and PPARγ.
Results. AP activity and calcium staining were both
increased at day 14 in the crude keratose (C-KOS), alphakeratose (A- KOS), and gamma-keratose (G-KOS) treated
samples compared to controls. qRT-PCR showed large
fold changes for differentiating hADSC, particularly for
gamma-keratose and crude keratose (which contains
gamma-keratose; Figure 1). Also, PPARγ gene expression
was down regulated in the presence of all keratose
fractions compared to osteogenic media alone at all time
points (Figure 2).
Conclusion. Keratose biomaterials have a measurable
effect on osteogenesis as evidenced by increase AP
activity, calcification, and expression of important
osteogenic genes, seemingly through a down regulation
of PPARγ expression. This finding suggests that keratosebased bone grafts may have particular efficacy in elderly
patients where adipogenesis of autologous MSC used for
therapy may be unwittingly favored over osteogenesis.
Disclosures Mark Van Dyke holds stock and is an officer in
the company, KeraNetics LLC, who has provided partial
funding for this research. Wake forest University Health
Sciences has a potential finan
Keywords. Adipose Derived Stem Cells, osteogenesis,
keratin biomaterials, PPARγ
(1.O4) ADIPOSE-DERIVED STEM CELLS (ASCS) FROM
ANATOMICALLY
DIFFERENT
SITES
DETERMINE
PHENOTYPE AND FUNCTION OF SCHWANN-LIKE CELLS
FOR PERIPHERAL NERVE REPAIR
Haycock J (1), Kaewkhaw R (1), Scutt A (1)
1. Sheffield University
Adipose-derived stem cells (ASCs) have gained
considerable interest as a source for deriving other
specific cell types including Schwann cells for treating
peripheral nerve injury. However, our hypothesis was
that the adipose donor site might influence the
differentiation potential of ASCs into Schwann cells,
which is presently unknown. This work therefore
investigated the differentiation of ASCs harvested from
different anatomical sites of: i) subcutaneous; ii)
perinephric; and iii) epididymal adipose tissue. We
demonstrated that although these cell types shared a
common multilineage differentiation potential and cell
surface markers, ASCs from anatomically different sites
differed in their Schwann cell phenotype and function in
stimulating neuronal differentiation in vitro. The upregulation of S100β, GFAP and p75NGFR was observed in
perinephrium-ASCs, while only the expression of S100β or
GFAP and p75NGFR was elevated in subcutaneous-ASCs
or epididymis-ASCs. Co-culture of ASCs with NG108-15
neuronal cells showed that differentiated ASCs from each
source stimulated neurite outgrowth, which was
significantly greater than undifferentiated ASCs. In
addition,
subcutaneous
and
perinephrium-ASCs
stimulated neurite extension and sprouting number more
effectively than epididymis-ASCs. High levels of BDNF and
NGF were detected in differentiated ASCs in the above
co-cultures, but levels of NT-3 were low. We found that
through functional blocking studies to BDNF and NGF that
complete abrogation resulted, suggesting a major role of
these two neurotrophins in particular for stimulating
neuronal cell differentiation. Thus, ASCs can be obtained
from different anatomical sites and this determines the
differentiation potential, Schwann cell like phenotype and
extent of function. In conclusion, this work supports the
potential of ASCs as an alternative cell source to primary
Schwann cells for the local delivery and treatment of
peripheral nerve injury.
Keywords. Adipose stem cell; Schwann cell; Nerve
(1.O5) INTERACTION BETWEEN SHEAR STRESS AND VEGF
IN THE INDUCTION OF ENDOTHELIAL DIFFERENTIATION
OF HUMAN ADIPOSE – DERIVED STEM CELLS
Colazzo F (1), Alrashed F (2), Sarachandra P (3), Chester
AH (3), Yacoub MH (3), Taylor PM (3)
1. IRCCS Policlinico S. Donato, via Morandi 30, 20097, S.
Donato Milanese, Milan, Italy; 2. King Saud University
College of Medicine, King Fahad Cardiac Center, Riyadh.
KSA; 3.Heart Science Centre, NHLI, Imperial College
London, Harefield, Middlesex, UB9 6JH, UK
Introduction. Adipose tissue represents an abundant and
accessible source of adult stem cells with the ability to
differentiate into endothelial cells for therapeutic
vascularisation and tissue engineering applications.
However none of the studies to date have been able to
demonstrate
differentiated
cells
displayed
a
comprehensive range of endothelial characteristics.
Herein we combine chemical and mechanical stimulation
to investigate the effects of vascular endothelial growth
factor (VEGF) and physiological shear stress in promoting
the differentiation of human adipose derived stem cells
(ADSCs) into endothelial cells.
Materials and Methods. ADSCs were isolated and
characterised by immunofluorescence and flow
cytometry. Endothelial differentiation was promoted by
culturing confluent cells in the presence of 2% foetal calf
serum and 50ng/ml VEGF under physiological shear stress
(12 dynes) for up to 14 days. Endothelial characteristics
were evaluated by immunofluorescence staining for
endothelial markers, analysis of acetylated–low density
lipoprotein (Ac-LDL) uptake and assessment of tubular
formation performed using an in vitro angiogenesis assay.
Results. Human ADSCs treated with VEGF and subjected
to shear stress expressed vWF, eNOS and FLT-1 after 7
days and CD31, FLK-1 and VE-cadherin after 14 days.
Treated cells also were able to incorporate Ac-LDL as well
as form tubular structures on matrigel, unlike control
cells. Untreated cells or cells only subject to shear stress
did not display any of the noted endothelial
characteristics.
Conclusion. Based on these results, we have
demonstrated that ADSCs subject to mechanical shear
stress and chemical stimulation with VEGF are able to
express a comprehensive range of endothelial markers.
Our differentiation protocol provides a more efficient
strategy to obtain endothelial-like cells for tissue
engineering based on autologous, mesenchymal stem
cells (MSCs).
This research was supported by IRCCS Policlinico San
Donato, King Saud University and Magdi Yacoub Institute
founding’s. Authors thank Dr Basim A. Matti M.D. (Harley
Street Clinic, London) for providing adipose tissue
samples.
Keywords. ADSCs, Endothelial Differentiation, VEGF,
shear stress
(1.O6) CO-CULTURE OF HUMAN PREADIPOZYTES AND
ENDOTHELIAL
PROGENITOR
CELLS
FOR
NEOVASCULARISATION
OF
TISSUE
ENGINEERED
ADIPOSE TISSUE
Strassburg S (1), Nienhüser H (1), Stark GB (1), TorioPadron N (1)
1. Department of Plastic Surgery, University Medical
Center Freiburg, Hugstetter Straße 55, 79106 Freiburg,
Germany
Introduction. Tissue engineering of adipose tissue suffers
from the major disadvantage of tissue resorption due to
an insufficient vascularisation. Thus, a novel strategy to
create vascular networks and enhance neovascularisation
of tissue engineered adipose tissue might be the coimplantation of preadipozytes with endothelial
progenitor cells (EPCs). Here, we investigate the effects of
co-culture of preadipozytes and EPCs on EPC sprout
formation in vitro and neovascularisation of implants in
vivo.
Material and Methods. Preadipozytes were isolated from
human fat tissue and EPCs from human peripheral blood.
In vitro, the angiogenic effects of preadipozytes on EPCs
were analysed in an EPC spheroid sprouting assay. In vivo,
investigations to determine the vascularisation of Fibrin
implants due to co-culture of preadipozytes and EPC
spheroids were performed in a chick embryo
chorioallantoic membrane (CAM) assay. After 8 days, the
neovascularisation of the implants were evaluated by
histological analyses.
Results. In vitro, co-culture with preadipozytes induces
significant longer sprout formation in EPCs compared to
EPC spheroids alone. In vivo, implants containing
preadipozytes and EPC spheroids displayed a significant
higher rate of neovascularisation in terms of number and
depth compared to preadipozytes or EPC spheroids alone
where less or no vessel ingrowth was observed.
Discussion. Co-culture of preadipozytes and EPCs
enhances the angiogenic capacities in vitro and in vivo.
Thus, this study highlights the importance of cellular
contact between preadipozytes and EPCs for
neovascularisation of tissue engineered adipose tissue.
Keywords. preadipozytes, endothelial progenitor cells,
co-culture, CAM, angiogenesis
(1.O7) ACCELERATION AND AUGMENTATION OF
FEMORAL SEGMENTAL BONE HEALING BY ADIPOSEDERIVED STEM CELLS ENGINEERED BY HYBRID
BACULOVIRUS VECTORS CONFERRING SUSTAINED
TRANSGENE EXPRESSION
Lin CY (1), Lin KJ (2), Kao CY (1), Chang YH (3), Hu YC (1)
1. National Tsing Hua University; 2. Chang Gung
University; 3. Chang Gung Memorial Hospital
Introduction. Massive segmental defects arising from
trauma or tumor resection remain a challenging clinical
problem. To heal massive, segmental bone defects using
adipose-derived stem cells (ASCs) which alone cannot
heal large defects, we hypothesized that sustained
expression of factors promoting bone regeneration
(BMP2) and angiogenesis (VEGF) provides continuous
stimuli to augment the healing. Baculovirus (BV) holds
promise for gene therapy and efficiently transduces stem
cells, but it only mediates transient transgene expression.
Materials and Methods. We developed a dual BV system
whereby one BV expressed FLP recombinase (BacFLP)
while the other hybrid BV harbored an Frt-flanking
transgene cassette for ASCs engineering and healing of
critical-size segmental bone defects in New Zealand
White (NZW) rabbits. Whether the ASCs persistently
expressing BMP2/VEGF expedited the healing was
assessed by X-ray, PET/CT, µCT, histochemical staining
and biomechanical testing.
Results. We confirmed that within ASCs transduced with
BacFLP and the hybrid BV, FLP/Frt-mediated
recombination occurred in up to 46% of ASCs, leading to
cassette excision off the BV genome, formation and
persistence of episomal transgene and prolongation of
expression to >28 days. Transduction of ASCs with the
BMP2-expressing hybrid BV prolonged the BMP2
expression and augmented osteogenesis of ASCs even
without osteogenic supplements. ASCs engineered by the
hybrid vectors mediating sustained BMP2/VEGF
expression healed the critical-size (10 mm) segmental
bone defects in 12 out of 12 rabbits in 8 weeks, which
remarkably outperform ASCs engineered with BV
transiently expressing BMP2/VEGF with respect to healing
rate, bone metabolism, bone volume, bone density,
angiogenesis and mechanical properties.
Conclusion. These data attested our hypothesis that
persistent BMP2/VEGF expression are essential when
using ASCs for repairing massive defects. The use of ASCs
engineered with the hybrid BV vector represents a novel
therapy to treat massive segmental defects necessitating
concerted ossification and vascularization.
Keywords. baculovirus, adipose-derived stem cells,
segmental bone defect, sustained expression
(1.O8) EVALUATION OF DIFFERENT SCAFFOLD DESIGNS
FOR VASCULARIZED ADIPOSE CONSTRUCTS
Wiggenhauser PS (1), Mueller DF (1), Hutmacher DW (2),
Melchels FPW (2), Storck K (1), Staudenmaier R (1),
Machens HG (1), Schantz JT (1)
1. Muenchen Rechts der Isar, Technische Universitaet
Muenchen; 2. Institute of Health and Biomedical
Innovation, Queensland University of Technology
Free fat grafts are frequently used in plastic and
reconstructive surgery to treat large volume defects e.g.
breast reconstruction. Current clinical limitations are
however in larger defects which need vascularized fat
grafts in order to improve the survival and in addition the
need to provide a 3D predictable structure. Recently
described innovative scaffold fabrication systems allow
patient specific scaffold fabrication and thus engineering
of
customized
fat
grafts.
We
investigated
Polycaprolactone (PCL) scaffolds made by fused
deposition modeling and Polyurethane (PU) sponges
made by solvent casting in a combined in vitro and in vivo
study. Scaffolds were evaluated in respect to adipose
tissue engineering.
Scaffold structure was analyzed with SEM and µCT.
Scaffolds were then seeded with human adipoderived
progenitor cells which were obtained from lipoaspirates.
Cell seeded constructs were cultured in adipogenic
culture media for 2 weeks and were analyzed
biochemically and microscopically. Subsequently the
constructs were implanted in nude mice for in vivo
studies. Femoral artery and vein were dissected and
placed upon constructs to mimic a vessel loop for
vascularization. Constructs were explanted after 2 and 4
weeks and histologically processed. Adipoderived
progenitor cells attached to both scaffolds and showed an
increase (p<0.05) of metabolic activity in experimental
groups. Formation of fat tissue was superior (p<0.05) in
PU-scaffolds compared to PCL-scaffolds in vitro and in
vivo. However vascularization of constructs was equal
within all groups.
In conclusion both scaffold systems represent suitable
carriers for adipose tissue formation in vitro and in vivo.
The advantage of rapid prototyping technology allows
production of customized vascularized grafts, which have
great potential especially for breast reconstruction.
Keywords. polycaprolactone, polyurethane, vascularized
adipose constructs, in vitro, in vivo
(1.O9) CARTILAGINOUS TISSUES ENGINEERED USING
HUMAN FAT PAD DERIVED MESENCHYMAL STEM CELLS
UNDER ALTERED DIFFERENTIATION CONDITIONS
Liu Y (1), Buckley CT (1), Downey R (2), Mulhall KJ (2),
Kelly DJ (1)
1. Trinity Centre for Bioengineering, School of Engineering,
Trinity College Dublin, Ireland; Sports Surgery Clinic,
Dublin, Ireland; 2. Sports Surgery Clinic, Dublin, Ireland
Introduction. Adult stem cells from adipose tissue can
potentially be used in cell-based therapies for cartilage
repair. Previous studies from our lab have shown that
functional cartilage tissue can be engineered using
porcine infrapatellar fat pad (FP) mesenchymal stem cells
(MSCs). It remains unclear if functional tissue can be
engineered using MSCs isolated from human
osteoarthritic FP tissue. The objective of this study was to
determine the influence of oxygen tension during
expansion and supplementation conditions during
differentiation on the functional properties of cartilage
tissues engineered using human FP MSCs.
Materials and Methods. Human infrapatellar FP was
harvested during total knee replacement following ethical
approval. The isolated FP MSCs was expanded under 21%
or 5% O2. After expansion to P2, MSCs was seeded in
agarose at 10 million cells/ml. Cell pellets (250,000
cells/pellet) were also cultured as a control. Both cellseeded agarose hydrogels and pellets were cultured at
5% pO2 with chondrogenic differentiation medium with
different supplements: (1) TGF-β3 (10ng/ml), (2) TGFβ3+2% fetal bovine serum (FBS), (3) TGF+10% FBS or (4)
TGF-β3+BMP-6 (10ng/ml). Constructs were analyzed
using DNA, GAG, and collagen assays.
Results. DNA content within the agarose hydrogels
increased with the supplement of TGF-β3+10%FBS, in
contrast to the reduced DNA content in other groups. The
addition of serum also promoted GAG and collagen
accumulation. In contrast, in pellet culture, only small
differences were observed between constructs
supplemented with different factors (data not shown).
Conclusion. Robust chondrogenic differentiation of
human FP derived MSCs was observed following agarose
encapsulation for constructs supplemented with TGFβ3+10%FBS. FP MSCs appear to respond differently to
media supplementation if cultured in hydrogels or pellets.
This work was supported by IRCSET / Sports Surgery Clinic
(Dublin) Enterprise Partnership Scheme, Science
Foundation Ireland and the European Research Council.
Keywords. human fat pad mesenchymal stem cells;
chondrogenesis; differentiation conditions
Conclusion. Adipogenesis in the spheroid system proved
to be less dependent on external stimulation than in
conventional 2D culture. The characterization of the 3D
spheroids provided valuable information for their use in
adipose tissue engineering as well as in basic research.
Keywords. adipose tissue engineering; adipose-derived
stem cells; adipogenesis; spheroids
(1.O10) ADIPOGENESIS IN 3D SPHEROIDS OF ADIPOSEDERIVED STEM CELLS IS LESS DEPENDENT ON
EXOGENOUS STIMULATION THAN IN CONVENTIONAL 2D
CULTURE
Muhr C (1), Winnefeld M (2), Pielmeier C (3), Seitz AK (3),
Göpferich A (3), Bauer-Kreisel P (4), Blunk T (4)
1. Department of Trauma, Hand, Plastic & Reconstructive
Surgery,
Julius-Maximilian-University,
Würzburg,
Germany; 2. Beiersdorf AG, Hamburg, Germany; 3.
Department of Pharmaceutical Technology, University of
Regensburg, Regensburg, Germany; 4.Department of
Trauma, Hand, Plastic & Reconstructive Surgery, JuliusMaximilian-University, Würzburg, Germany
Introduction. 3-dimensional (3D) spheroids of human
adipose-derived stem cells (hASC) have the potential to
serve as building blocks for adipose tissue engineering.
They also constitute an alternative model system for basic
research, allowing investigations of cellular processes in a
more in vivo-like context, representing cell-cellinteractions and the influence of the extracellular matrix
more closely than conventional 2-dimensional (2D)
culture.
Materials and Methods. Using hASC, a 3D carrier-free
spheroid model of human adipose tissue was established
utilizing the liquid overlay technique. Characterizing the
3D culture system, differences in the process of
adipogenesis between 3D spheroids and 2D culture were
addressed on a functional and molecular level by
investigating lipid accumulation and gene expression
(TaqMan® array).
Results. Applying short-term adipogenic induction
(common hormonal cocktail for two days), a strong
adipogenic response with a high lipid content on day 14
was observed in 3D spheroids, whereas lipid content was
only minimal in 2D culture. Gene expression data
reflected these results: In 2D culture, several genes
associated with lipid synthesis and transport (FASN, ACLY,
FATP1) were very weakly expressed, in contrast to high
expression in 3D spheroids. Also other fat cell markers
and adipokines (e.g., adiponectin, apelin, LPL) were more
strongly expressed in 3D. Strikingly, already on day 2,
increased expression of important transcription factors
(PPARγ, C/EBPβ, SREBF1) was determined in 3D culture,
which represents, at least in part, a likely explanation for
the observed 2D/3D-differences at later time points. In
2D, further exogenous stimulation after day 2 was
necessary
to
achieve
significant
adipogenic
differentiation, while the 3D context provided conditions
rendering further stimulation unnecessary.
(1.O11) 5-AMINOSALICYLIC ACID TO SUPPORT
ADIPOGENIC DIFFERENTIATION OF ADIPOSE TISSUE
DERIVED STEM CELLS IN 2 AND 3-D CULTURES
Manhardt M (1), Ambrosch K (1), Hacker MC (1), SchulzSiegmund MB (1)
1. Pharmaceutical Technology, Institute for Pharmacy,
University of Leipzig
Introduction. The aim of this study was to develop a
suitable protocol for adipogenic differentiation of rat
adipose tissue stem cells (ADSCs) and to investigate its
use in different 3-D culture systems. This protocol
involved standard supplements for adipogenic induction
added for 4 days and supplementation of 5-aminosalicylic
acid (5-ASA) with 2% FBS thereafter. Using indomethacin,
rosiglitazone and celecoxib as alternative to 5-ASA, we
tried to shed light on the mechanisms of 5-ASA. Three
different scaffold systems, made from either PLGA or PCL
served to find suitable 3-D systems for tissue
development.
Materials and Methods. Proliferation: in DMEM high
glucose, 10% FBS and 1% PenStrep with 3 ng/ml bFGF,
seeding on scaffold systems: centrifugation method and
proliferation for another 5 days with bFGF-supplemented
medium. Induction: 4 days insulin, dexamethasone, IBMX
and indomethacin in proliferation medium without bFGF.
Maturation: unsupplemented basal medium (control) or
medium containing 0.3 mM 5-ASA. Characterization of
adipogenic development on different scaffolds: protein
levels, glycerol-3-phosphate-dehydrogenase (GPDH)
activity and triglyceride content. Staining of cells: DAPI,
Nile Red and osmium tetroxide for light, fluorescence
microscopy and SEM imaging after lyophilisation.
Results and Conclusion. The new protocol involving 5ASA and reduced FBS in 2- and 3-D led to improved
adipogenic differentiation compared with continuous
supplementation of induction cocktail and control after
only 8 days of adipogenic stimulation. Groups receiving 5ASA or celecoxib differentiated better than groups
treated with indomethacin or rosiglitazone, indicating
COX-2 involvement in the adipogenic effect. ADSCs
attached and proliferated well on all three investigated
scaffold types. Differentiation, however, was weak on
electrospun PCL fibers compared to PLGA scaffolds and
microscaffolds with larger pore sizes. The results show
that the new protocol promotes adipogenic
differentiation of ADSCs on 3-D carriers in the presence of
reduced FBS and 5-ASA. In contrast to standard protocols
these conditions can be generated in vivo.
Keywords. bFGF, rat ADSC, polymer scaffolds, Mesalazin,
5-aminosalicylic acid
(1.O12) IN VITRO EVALUATION OF OSTEOCONDUCTIVE
STARCH BASED SCAFFOLDS USING A FLOW PERFUSION
BIOREACTOR
Rodrigues AI (1), Costa P (1), Gomes ME (1), Leonor IB (1),
Reis RL (1)
1. 3B’s Research Group – Biomaterials, Biodegradables
and Biomimetics, University of Minho, Portugal
Introduction. This works aims at studying the potential of
SPCL wet-spun fiber-meshes functionalized with silanol
groups as a bioactive matrix enabling highly tailored
cellular environments and thus promoting osteogenic
differentiation in human adipose stem cells (hASCs).
Another point of interest in this work is to understand the
influence of a dynamic culture, particularly using a flow
perfusion bioreactor, in hASCS cultured onto the
functionalized materials.
Materials and Methods. The functionalization of the
materials was achieved by a one step methodology using
a calcium silicate solution as a coagulation bath for fiber
meshes production. After an optimization on the
production procedure of the materials, some conditions
were selected for biological assays. The influence of the
presence of silicium ions in the material, along with a
dynamic culturing, on the adhesion, differentiation and
proliferation of hASCs was assessed.
Results. The functionalized materials exhibit the capacity
to sustain cell proliferation and induce their
differentiation into the osteogenic lineage. The formation
of mineralization nodules was observed in cells cultured
onto the functionalized materials. The culturing under
dynamic conditions by using a flow perfusion bioreactor
was shown to enhance hASCs proliferation and
differentiation and a better distribution of cells within the
material.
Conclusion. The promising properties of the
functionalized materials along with a simple, economic
and reliable production process demonstrate the
potential of these materials as candidates for application
in bone tissue engineering. The culture of stem cells onto
these materials using a flow perfusion bioreactor reveals
to be a good strategy to promote osteogenic
differentiation.
This work was supported by the European NoE
EXPERTISSUES (NMP3-CT-2004-500283) and by the
Portuguese Foundation for Science and Technology, FCT,
through the projects PTDC/CTM/67560/2006. I. B. Leonor
thanks the Portuguese Foundation for Science and
Technology (FCT) for providing her a post-doctoral
scholarship (SFRH/BPD/26648/2006).
Keywords. Bone tissue engineering, adipose stem cells,
silanol groups, instructive materials, wet-spinning, flow
perfusion bioreactor.
(1.O13) CYCLIC UNIAXIAL STRAIN UPREGULATES THE
SKELETAL MUSCLE-RELATED GENES IN ADIPOSE-DERIVED
STEM CELLS
Bayati V (1), Sadeghi Y (1), Shokrgozar MA (2),
Haghighipour N (2)
1. Shaheed Beheshti University of Medical Sciences; 2.
National Cell Bank of Iran, Pasteur Institute of Iran
Introduction. It has been revealed that skeletal muscles
have the potential to generate and respond to
biomechanical signals and that the mechanical force is
one of the important factors that influence proliferation,
differentiation, regeneration and homeostasis of skeletal
muscle and myoblasts. The aim of our study was to
illustrate the role of cyclical strain on myogenic
differentiation of adipose-derived stem cells (ASCs).
Materials and Methods. we designed a study within
three days with 3 groups: chemical, chemical-mechanical,
mechanical on the basis of stimulation of ASCs with
chemical factors (on the whole three days) or mechanical
strain (just on the second day) and compared the relative
expression of myogenic-related genes MyoD, Myogenin
and myosin heavy chain 2 (MyHC2) with expression of the
same genes in undifferentiated ASCs by Relative gene
expression method.
Results. Real-time RT-PCR results demonstrated that
uniaxial strain had a significant effect on up-regulation of
muscle-related genes in chemical-mechanical group
(P<0.05) compared to mechanical or chemical groups.
Immunocytochemistry also confirmed the myogenic
differential effect of cyclic strain on ASCs and showed that
this also influenced ASCs morphology and their
orientation.
Conclusion. These data suggest that uniaxial cyclic strain
could possibly affect the myogenic differentiation of ASCs
and cause the muscle-related genes to increase beyond
their basal level in ASCs and that the combination of
chemical myogenic approach with mechanical signals
promote differentiation more than differentiation by
chemical approach alone
Keywords. uniaxial cyclic strain, adipose-derived stem
cells, skeletal myogenic differentiation
(1.O14) ENHANCED CARTILAGE FORMATION VIA THREEDIMENSIONAL ENGINEERING OF HUMAN ADIPOSEDERIVED STROMAL CELLS
Yoon HH (1), Bhang SH (1), Shin JY (1), Shin JH (1), Kim BS
(1)
1. School of Chemical and Biological Engineering, Seoul
National University, Seoul 151-744, Republic of Korea
Introduction. Damaged articular cartilage has poor
intrinsic regenerative capacity. Autologous chondrocyte
transplantation is an effective treatment but involves
surgical procedures which may cause further cartilage
degeneration. Additionally, in vitro expansion of
chondrocytes can result in dedifferentiation and
phenotypic property loss. Human adipose-derived stem
cells (hADSCs) are an alternative autologous cell source
for cartilage regeneration due to their multipotency,
relatively easy accessibility and expansion. In this study,
we developed an efficient method for in vitro
chondrogenic differentiation of hADSCs and in vivo
cartilage formation of hADSCs and elucidated the
mechanisms of the enhanced in vitro chondrogenesis.
Materials and Methods. In vitro chondrogenesis of
hADSCs was promoted by culturing hADSCs in spheroid
form in spinner flasks. As a control, hADSCs were cultured
in monolayers in tissue culture dishes. Signaling cascades
for chondrogenesis of hADSCs cultured with two different
methods were examined. To evaluate in vivo cartilage
forming ability of the cells, hADSCs cultured either in
spheroid form or in monolayers were mixed with fibrin
gel and implanted subcutaneously into athymic mice for
four weeks.
Results. Polymerase chain reaction (PCR), quantitative
real-time
(qRT)-PCR,
and
immunohistochemistry
indicated enhanced chondrogenic differentiation of
hADSCs cultured in spheroid forms versus those cultured
in monolayers. The enhanced chondrogenesis is likely
attributed to mild hypoxia-related cascades and
enhanced cell-cell interactions of hADSC spheroids. The in
vivo study showed enhanced cartilage formation by
implantation of spheroid-cultured hADSCs versus
monolayer-cultured hADSCs.
Conclusion. Spheroid culture in three-dimensional
bioreactors is advantageous over monolayer culture for in
vitro chondrogenic differentiation of hADSCs and
subsequent in vivo cartilage formation.
This study was funded by grant (2010-0020352) from the
National Research Foundation of Korea
Keywords. human adipose-derived stromal cells,
spheroids, three dimensional culture, cartilage formation
(1.O15) RETRO-ASSOCIATED VIRAL GENE TRANSFER OF
SOX-TRIO TO HUMAN BONE MARROW DERIVED
MESENCHYMAL STEM CELLS IMPROVES CARTILAGE
REPAIR
Lee JS (1), Kim HJ (1), Im GI (1)
1. Dongguk International Hospital, Republic of Korea
Objective. The aim of this study was to test the
hypotheses that retroviral gene transfer of SOX trio
enhances the in vitro chondrogenic differentiation of
ASCs, and that SOX trio-co-transduced ASCs promote the
healing of osteochondral defects, and arrest the
progression of surgically-induced osteoarthritis in a rat
model.
Materials and Methods. ASCs isolated from inguinal fat in
rats were transduced with SOX trio genes using
retrovirus, and further cultured in vitro in pellets for 21
days, then analyzed for gene and protein expression of
SOX trio and chondrogenic markers. Sox trio-cotransduced ASCs were implanted on the osteochondral
defect created in the patellar groove of the distal femur,
and also injected into the knee joints of rats with
surgically-induced osteoarthritis. Rats were sacrificed
after 8 weeks, and analyzed grossly and microscopically.
Results. After 21 days, ASCs transfected with a single
gene of the SOX trio had a 140 to 320-fold greater gene
expression of SOX-5, -6, or -9 compared with the control
while ASCs co-transfected with SOX trio had 40 to 70-fold
greater gene expression. The SOX protein expression
paralleled that of gene expression. The GAG content
increased approximately 6-fold with SOX trio cotransduction. SOX trio co-transduction significantly
increased type II collagen gene and protein expression.
SOX trio co-transduction significantly promoted cartilage
healing in the in vivo osteochondral defect model, and
prevented the progression of degenerative changes in
surgically-induced osteoarthritis.
Conclusion. SOX trio co-transduction enhances
chondrogenesis from ASCs. SOX trio-co-transduced ASCs
promote healing of cartilage defects and arrest the
progression of osteoarthritis.
This work was supported by a grant from the Korea
Ministry of Education, Science and Technology (Grant No
2010-0000305).
Keywords. retrovirus
(1.O16) THE USE OF STEM CELL CULTURE-CONDITIONED
MEDIUM FOR THERAPEUTIC ANGIOGENESIS
Bhang SH (1), Kim BS (1)
(1) Seoul National University, Republic of Korea
Introduction. Stem cell implantation can be used to
induce neovascularization and has been tested as a
therapy for ischemia treatment. However, stem cell
implantation as a therapy for ischemia treatment may
have limitations for clinical applications. Since the
methods of stem cell harvest are invasive, it may not be
feasible to harvest autologous stem cells from aged
patients or patients with cardiovascular risk.
Furthermore, poor cell survival after engraftment in
ischemic tissue may lower the therapeutic efficacy of
stem cells. hADSCs implanted to ischemic tissues support
tissue revascularization in large part through secreted
angiogenic factors. The goal of this study is to
demonstrate that medium collected from human
adipose-derived stromal cells (hADSCs) cultured as
spheroids can exhibit improved therapeutic efficacy for
ischemia treatment.
Materials and Methods. Conditioned medium derived
from hADSC monolayer culture (M-CM) or spheroid
culture (S-CM), fresh medium (FM), or hADSCs were
injected intramuscularly into the gracilis muscle in the
medial thigh after mouse hindlimb ischemia modeling.
Results. Due to a mild hypoxic environment formed in
hADSC spheroid, spheroid culture was effective to
precondition the hADSCs to upregulate hypoxia-inducible
factor-1α gene expression following significant
enhancement in both angiogenic and anti-apoptotic
factor secretion to the culture medium compared to
monolayer cultures. S-CM administration to ischemic
hindlimbs
in
mice
significantly
enhanced
neovasclurization, protected muscles from incipient
ischemic apoptosis, and improved limb survival as
compared to M-CM or FM administration or hADSC
implantation.
Conclusions. These data suggest that injection of
conditioned medium obtained from hADSC spheroid
culture may be more effective therapeutic option for
treatment of ischemic diseases than hADSC implantation.
Keywords. angiogenesis, stem cell, conditioned medium
(1.O17)
PHARMACOLOGICAL
MODULATION
OF
MESENCHYMAL STEM CELL CHONDROGENESIS BY
MARINE POLYSACCHARIDES FOR CARTILAGE TISSUE
ENGINEERING
Merceron C (1,2), Rederstorff E (1,3), Portron S (1,2),
Colombeix C (1,2), Masson M (1,2), Lesoeur J (1,2),
Sourice S(1,2), Colliec-Jouault S (3), Weiss P (1,2), Vinatier
C (1,2,4) Guicheux J (1,2)
1. INSERM (Institut National de la Santé et de la
Recherche Médicale), UMRS 791, Université de Nantes,
Laboratoire d'Ingénierie Ostéo-Articulaire et Dentaire,
Group STEP “Skeletal Tissue Engineering and
Physiopathology”, Faculté de chirurgie dentaire, Nantes,
France; 2. PRES-UNAM, UFR Odontologie, Université de
Nantes, France; 3. Laboratoire de biotechnologie et
molécules marines (BRM/BMM), Ifremer, Nantes, France ;
4. Graftys SA Aix en Provence, France.
Mesenchymal stem cells (MSC) are considered as an
attractive source of cells for cartilage engineering owing
to their availability, capacity of in vitro expansion and
multipotency. Differentiation of MSC into chondrocytes is
crucial to successful cartilage regeneration and can be
induced by a large variety of biological agents and
environmental factors. Glycosaminoglycans (GAGs) are
complex carbohydrates that participate in many biological
processes through interaction with various proteins
including growth factors. We hypothesize that growth
factors-induced differentiation of mesenchymal stem
cells could be potentiated by marine polysaccharides.
To test our hypothesis MSC were isolated from human
adipose tissue obtained by liposuction. Human adipose
tissue derived MSC (hATSC) were cultured three
dimensionally in pellets in the presence of TGF-supplemented chondrogenic medium containing or not
two marine polysaccharides analogs of low molecular
weight (LMW1 and LMW2, patenting in progress).
Chondrogenesis was monitored by the measurement of
pellet volume and histological stainings (Alcian blue and
hematoxylin) of the pellets. Our data revealed an increase
in pellet volume as well as in total collagens and GAG
production in the concomitant presence of LMW1 (and
not LMW2) and chondrogenic medium. The enhanced
hATSC chondrogenesis in response to LMW1 treatment
was further demonstrated by the increased expression of
COL2A1, ACAN, COMP and SOX9 by real time PCR. In
addition, surface plasmon resonance (Biacore) analyses
revealed that TGF-β1, but not insulin, binds LMW1 with
higher affinity compared to LMW2 polysaccharide.
Furthermore LMW1 marine polysaccharide was found to
up-regulate the TGF-β dependent phosphorylation of
ERK1/2, indicating that LMW1 marine polysaccharide
enhanced the MAP kinase signaling activity of TGF-β.
These results demonstrate the up-regulation of the TGFbeta-dependent stem cell chondrogenesis by a marine
polysaccharide. Whether this data may help monitor and
exploit the potential of MSC for cartilage regeneration
would be paid further attention.
Keywords. adipose tissue derived stem cells, cartilage
tissue
engineering,
marine
polysaccharide,
glycosaminoglycan-mimetic
(1.O18) HUMAN ADIPOSE DERIVED STROMAL CELL
RESPONSE TO A POLY ε-CAPROLACTONE SCAFFOLD FOR
BONE TISSUE ENGINEERING
Pagani S (1) , Veronesi F (1), Parrilli A (1), Maltarello MC
(1), Salerno A (2), Fini M (1), Giavaresi G (1)
1. Laboratory of Preclinical and Surgical Studies, Rizzoli
Orthopaedic Institute - IRCCS, Bologna, Italy; 2.
Interdisciplinary Research Centre on Biomaterials CRIB,
Naples, Italy
Introduction. Tissue engineering represents an
interesting challenge to heal several bone lack. The
adipose tissue, normally discarded during plastic surgery,
has been demonstrated to be an alternative source of
stromal cells. The aim of the present study was to
evaluate the ability of porous poly(ε-caprolactone) (PCL)
scaffold with novel bimodal-micron scale porous
architecture (µ-bimodal PCL), to promote and guide the in
vitro adhesion, proliferation and 3D colonization of
human adipose derived stromal cells(hADSCs).
Materials and Methods. The µ-bimodal PCL scaffold was
prepared by the combination of the gas foaming abd
selective polymer extraction from co-continous blends
techniques. Human ADSCs were enzimatically isolated
from fat collected during lipectomy and their
characterization was assessed by flow cytometry. Then,
hADSCs were expanded and seeded on µ-bimodal PCL
scaffolds with an osteogenic medium. Cell adhesion
(SEM), proliferation (Pico Green) and viability (Alamar
blue), osteoblast differentiation (ALP) and 3D scaffold
colonization were assessed at 24h, 1 and 2 weeks. In
particular, 3D scaffold colonization was evaluated by
using SKYSCAN 1172 microtomographer (µCT).
Results. hADSC resulted positive for CD44, CD73, CD90
and CD105. After 24h, SEM showed that hADSC cell
adhered and entirely colonized the seeded surface of PCL
scaffold. Cell viability and proliferation increased
significantly over experimental time. µCT showed that
hADSCs uniformly colonized the entire thickness of the
scaffold.
Conclusion. The µ-bimodal PCL/hADSCs interaction study
showed the ability of the scaffold to support hADSCs
adhesion and proliferation, as well as to promote and
guide 3D cell colonization by appropriately designing the
microarchitectural features of the scaffold. At the same
time, the opportunity of using a novel, non-invasive
method as µCT makes easier and more accurate the
analysis of the construct in vitro, above all in respect to
the cellular distribution.
Keywords. poly(ε-caprolactone), human adipose derived
stromal cells
(1.P1) DEVELOPMENT OF A PROTOCOL FOR HUMAN
ADIPOSE STEM CELL CULTURE IN CO2 INDEPENDENT
MEDIUM IN PERFUSION BIOREACTOR
Silva ARP (1), Paula ACC (1), Zonari AAC (1), Martins TM
(1), Goes AM (1), Pereira MM (1)
1. UFMG, Brazil
Advances in research on stem cells derived from human
adipose tissue (hASC) may allow its use for cell therapy
and tissue engineering. In such context, it is important to
standardize a methodology to culture cells in high
quantity. Bioreactors, in which cells are cultured in threedimensions and may use CO2 independent media, mimic
the physiological environment in vitro, allowing the hASC
proliferation, differentiation and maintenance. In
addition to the cells and the cell culture medium, a
suitable biomaterial is critical to the success of bone
tissue regeneration. In this study, a sol-gel bioactive glass
(BG) was the material of choice, due to its osteoinductive
properties. The aim of the study was to evaluate
phenotypic stability, proliferation, cell viability and
protein secretion by hASC cultured in CO2 independent
medium in three-dimensional cell culture in perfusion
bioreactor. The hASC was isolated from human
lipoaspirate and two-dimensional cell culture was
performed in DMEM supplemented with 10% FBS. The
cellular adaptation from DMEM to Leibovitz's CO2
independent medium supplemented with 10% FBS (Lei)
was gradual, beginning in the first passage with 25%Lei,
the second passage with 50%Lei, the third passage with
75%Lei and the fourth passage with 100%Lei. Phenotypic
characterization was performed by flow cytometry
analysis of the following markers: CD29, CD44, CD73,
CD34, CD45, HLA-ABC and HLA-DR. Cell proliferation and
viability in BG were evaluated by MTT assay. The
undifferentiated state was assessed by Alkaline
Phosphatase Activity assay of cells cultured in two- and
three-dimensions in Lei at 7, 14 and 21 days. Twodimensional comparative tests were performed in DMEM
as control. The results suggest that the Lei CO2
independent medium may be a promising model for in
vitro expansion of hASC for use in perfusion bioreactor.
The authors gratefully acknowledge the financial support
from CNPq and FAPEMIG/Brazil.
Keywords. hASC, Bioactive Glass, Bioreactor, CO2
Independent Medium
(1.P2) PIG MANDIBULAR RECONSTRUCTION BY ADIPOSEDERIVED STEM CELLS AND FUNCTIONALIZED LASERSINTERED POROUS PCL SCAFFOLD WITH PLATELET RICH
PLASMA: IN-VITRO AND IN-VIVO STUDY
Tsung LH (1), Chen JP (2), Lee MY (3)
1. Department of Plastic and Reconstructive Surgery,
Chang Gung Memorial Hospital, Chang Gung University;
2. Department of Chemical and Materials Engineering,
Chang Gung University; 3. Department of Mechanical
Engineering, Chang Gung University
Introduction. Polycaprolactone (PCL) is a bioresorbable
polymer with potential applications for bone and
cartilage repair. In this work, the three-dimentional and
porous PCL scaffolds were designed and it was fabricated
via selective laser sintering (SLS). The aim of this study is
to evaluate the osteogenic potential of adipose-derived
stem cells (ASCs) in functionalized laser-sintered PCL
scaffold with platelet rich plasma (PRP).
Materials and Methods. In the in-vitro study, the laser–
sintered PCL scaffold was seeded with ASCs. It was
divided into three groups. Group I: PCL/ASCs were
cultured in control medium. Group II: PCL/ASCs were
cultured in osteogenic medium. Group III: PCL/PRP/ASCs
were cultured in osteogenic medium. Alkaline
phosphatase activity, RT-PCR of ALP, osteocalcin, RunX II
were used to assess the osteogenic ability. SEM and
confocal microscope were used to observe the interaction
between scaffold and cell. In In-vivo study, the 3 cm
porcine mandible defect was created and it was
reconstructed with either PCL only or PCL/PRP/ASCs. CT
was used to evaluate the bone regeneration 3 months, 6
months after operation. The Young’s modulus of both
groups was measured and compared with normal bone.
H&E stain and IHC stain of osteocalcin, collagen type I
were done for confirmation of bone regeneration.
Results. In in-vitro study, alkaline phosphatase activity
and RT-PCR all showed the best osteogenic potential in
group III(PCL/PRP/ASCs) comparing with other groups.
SEM and confocal microscope showed the cells were well
attached to PCL in group III. All these data confirms that
the PCL combined with PRP was suitable for osteogenic
differentiation and attachment of ASCs. In in-vivo study,
both groups showed new bone regeneration in PCL
scaffold. However, the bone density was less and loose in
PCL group and the Young’s modulus was only 30% of
normal bone. In contrast, the continual and firm bone
formation was found in PCL/PRP/ ASCs gorup and the
Young’s modulus was 90% of normal bone. H&E stain, IHC
of osteocalcin, collagen type I all proved the new
generation tissue was bone.
Conclusion. In conclusions, modification of the lasersintered PCL scaffold by PRP enhances the affinity and
osteogenic potential of ASCs.
Keywords. selective laser sintering, Polycaprolactone,
platelet rich plasma, adipose derived stem cell
(1.P3) INFLUENCE OF SUBSTRATE’S RIGIDITY ON
ADIPOSE DERIVED STEM CELLS DIFFERENTIATION
Walenko K (1), Witkowska-Zimny M (1), LewandowskaSzumiel M (1)
1. Department of Biophysics and Human Physiology,
Medical University of Warsaw, Poland
Introduction. In 2006 intriguing data published in Cell
showed the significance of the stiffness of cell support for
stem cell differentiation. Since then, a few new reports
have revealed the influence of substrate rigidity on cell
morphology, motility or viability, however no new data
on cell differentiation are available. Since it might have a
practical implications for tissue engineering, in this work
we analyzed the response of human adipose derived stem
cells (HASCs) toward substrate elasticity with particular
attention paid to osteogenic differentiation.
Materials and Methods. HASCs from three donors (each
population in a separate experiment) were observed in a
culture for 14 and 21 days. Inert polyacrylamide gels
(PAAM) of two different rigidities (Young Modulus: 2,6kPa
and 28,1kPa) served as a support. Cell adhesion was
enhanced by coating the gels and control wells (TCPTissue Culture Plate) with collagen I. Both cell viability
(XTT-assay) and cell number (DNA determined in
PicoGreen) were assessed. Differentiation potential was
determined by measuring alkaline phosphatase (ALP)
activity and expression of ALP and RUNX2, as osteogenic
differentiation markers (real-time PCR).
Results. The support had no significant influence on cell
number or viability . HASCs differentiation confirmed by
RUNX2 and ALP expression was detected/observed in all
groups. RUNX2 expression was higher (1,5 fold) on more
rigid substrates as compared to the softer ones, either on
day 14 or 21 depending on the donor. ALP expression was
significantly higher on more rigid gels in all groups both
on 14 and 21 day (1,2-1,5 fold). This was accompanied by
the enhanced ALP activity on day 14 (1,9-5,3 fold), but
not on day 21.
Conclusion. We propose that HASCs, as cells with a high
rate of stemness are sensitive to the rigidity of the
support. The osteogenic differentiation is more advanced
on stiffer substrates.
Keywords. Adipose derived stem cells, elasticity,
differentiation
(1.P4) ISOLATION AND CHARACTERIZATION OF
MESENCHYMAL STEM CELLS FROM THE FAT LAYER ON
THE DENSITY GRADIENT SEPARATED BONE MARROW
Insausti, C (1), Blanquer, M (1), Meseguer L (1), Férez X
(1), Rodríguez, F (1), Cabañas, V (1), Funes C (1), Nicolás FJ
(2), Majado MJ (1), Moraleda JM (3)
1. Unidad de Terapia Celular. Servicio de Hematología.
HUVA, Murcia, Spain; 2. Unidad de Investigación. HUVA;
3. Universidad de Murcia, Murcia, Spain
Introduction. Bone marrow (BM) is considered the most
reliable source of adults MSCs. From this tissue MSCs can
be isolated after density gradient separation (ficoll) and
culturing the mononuclear cell fraction held in the
plasma-solution inter-phase at a density between 1.053
and 1.077, which is traditionally considered the only
source of progenitor cells (hematopoietic and nonhematopoietic). In this study we presented evidences that
MSCs could be also isolated from the very low-density
cells of the fat layer, normally discarded.
Material and Methods. BM aspirates were collected from
nine volunteers (6 males, 3 females, median age 26 years
old, range 6 to 45), after informed consent according to
the Hospital Ethic Committee. Samples were separated in
different fractions by ficoll density gradient method.
MNCs obtained from the plasma-solution inter-phase and
the very low density cells of the fat layer were collected,
counted and comparatively evaluated in primary cultures,
proliferation assays, ex vivo expansions, colony-forming
units-fibroblast tests, fluorescence activated cell sorting
analysis, and in vitro cell differentiation assays
Results. Cells coming from fat layer exhibited similar
proliferation characteristics than cells from the plasmasolution inter-phase. Colony-forming units-fibroblast
assays revealed similar efficiency. Proliferation rates of
MSCs from both sources were similar and so were the exvivo expansions. Immunophenotypical characterization of
MSCs showed similar antigens pattern. In vitro MSCs
differentiation potential was similar in both source cells.
Conclusion. MSCs could be isolated from the very lowdensity cells of the fat layer as from the MNCs at plasmasolution inter-phase. MSCs obtained from these cells have
similar characters than those obtained from the MNCs at
plasma-solution inter-phase. The method represents a
simple and cost effective way to increase the MSCs yield
from each BM donor. These cells might serve as a
complementary source of MSCs to facilitate preclinical
and clinical application in tissue engineering and cell
therapy.
Keywords. Mesenchymal Stem cells (MSCs), fat floating
cells, bone marrow, cell therapy, density gradient method
(1.P5) OSTEOGENIC TISSUE ENGINEERING BY ADIPOSE
TISSUE-DERIVED STEM CELLS IN VITRO
Peters K (1), Adam S (1), Salamon A (1), Neumann HG (2),
Rychly J (1), Kamp G (3)
1. Department of Cell Biology, Medical Faculty, University
of Rostock (Germany); 2. DOT GmbH, Charles-Darwin-Ring
1a, 18059 Rostock (Germany); 3. AMP-Lab GmbH,
Becherweg 9-11, 55099 Mainz (Germany)
Introduction. Adipose tissue-derived stem cells (ASC) are
able to differentiate along the osteogenic lineage, among
others. Since adipose tissue is an abundant source of
stem cells, tissue engineering approaches based on the
utilization of ASC are under development. In this study we
have examined in which way osteogenic differentiation of
ASC is affected by the supplementation with different
osteogenic factors and by 2- and 3-dimensional growth.
Materials and Methods. ASC cultivation was with DMEM,
10%
FCS
and
antibiotics
(basis
medium,
unstimulated/US). Osteogenic stimulation was with basis
medium supplemented with dexamethasone, ascorbic
acid, dinatriumglycerol-2-phosphate (OS) and/or BMP2.
Cultivation took place on tissue culture polystyrene
(TCPS) for 2D or type I collagen scaffolds for 3D
evaluation.
Results. Osteogenically stimulated ASC showed an
increase in cell number. In contrast, stimulation of ASC
with BMP2 led to a reduction. Addition of osteogenic
stimuli did not neutralize the effects of BMP2 on cell
number. Adhesion of ASC on TCPS and stimulation with
BMP2 induced spheroid formation (Fig.1a). On collagen
scaffolds, however, ASC developed a spindle-shaped
phenotype and no spheroid formation (Fig.1b). As
measured by alkaline phosphatase activity and extent of
mineralization, collagen scaffolds led to a higher cell
number and a higher degree of osteogenic differentiation
than TCPS. Fig. 1: BMP2-stimulated ASC on a) TCPS and b)
collagen scaffold (vital stain).
Conclusion. Thus, both ways of stimulation, i.e.
dexamethasoneand
BMP2-based,
affect
cell
proliferation and osteogenic differentiation. However, the
way of stimulation greatly changed other parameters of
cellular behavior. Growth on collagen scaffolds led to a
higher degree of osteogenic differentiation. Since stem
cells from adipose tissue are promising candidates for
tissue engineering approaches, further studies on the
mechanisms and reliability of osteogenic differentiation
of ASC are necessary.
This work was supported by the BMBF and the Federal
State of Mecklenburg-Vorpommern.
Keywords. adipose tissue-derived stem cells, BMP2,
collagen scaffold, osteogenic differentiation, tissue
engineering
(1.P6) CHONDROGENIC DIFFERENTIATION OF HUMAN
ADIPOSE TISSUE-DERIVED STEM CELLS ON GELATINBASED HYDROGELS IN VITRO
Salamon A (1), van Vlierberghe S (2), Adam S (1), Lochner
K (3), Bader R (3), Neumann HG (4), Rychly J (2), Dubruel P
(1), Peters K (1)
1. Department of Cell Biology, Medical Faculty, University
of Rostock; 2. Polymer Chemistry & Biomaterials Research
Group, Ghent University; 3. Department of Orthopaedics,
Medical Faculty, University of Rostock; 4. DOT GmbH,
Charles-Darwin-Ring 1a, 18059 Rostock
Introduction. Aim of this work was to characterize the
chondrogenic potential of human mesenchymal stem
cells from adipose tissue (ASC) in dependence on
adhesion to tissue culture polystyrene (TCPS) and gelatin
type B-based hydrogels. Thus, applicability of ASC for
chondrogenic tissue engineering approaches was
examined.
Materials and Methods. ASC were seeded in DMEM (10
% FCS) on 2.5 mm thick hydrogel films using
methacrylamide-modified gelatin type B (10w/v%) and on
TCPS. Chondrogenic stimulation was with ascorbic acid,
rhIGF-I, TGF-β1, ITS™, Dexamethasone and antibiotics in
serum-free medium. Gene expression of SOX5, SOX9,
COLI, and COLII was determined by real time PCR. Proof
of glycosaminoglycan synthesis was done by Alcian blue
staining.
Results and conclusion. ASC phenotypes differed clearly
in
dependency
on
the
adhesion
substrate:
chondrogenically stimulated ASC on TCPS showed a
cobblestone-like phenotype (Fig. 1a), whereas
chondrogenically stimulated ASC on hydrogels developed
spheroidal growth (Fig. 1b). Glycosaminoglycan synthesis,
a cartilage characteristic, was detected within the
hydrogel-induced spheroids. Chondrogenically stimulated
ASC on TCPS, however, were almost negative for
glycosaminoglycans.
Figure
1:
Chondrogenically
stimulated ASC on a) TCPS and b) hydrogels. Furthermore,
chondrogenic differentiation capacity of ASC was
examined by characterisation of cartilage-specific gene
expression. Therefore expression of chondrogenesisregulating factors was quantified. After chondrogenic
stimulation SOX5 was generally upregulated compared to
unstimulated ASC. The adhesion substrate (i.e. TCPS and
hydrogel) did not alter SOX5 gene expression. Expression
of COLI was not clearly regulated by chondrogenic
differentiation and adhesion substrate, whereas
expression of COLII was significantly increased by
chondrogenic stimulation of ASC in contact to hydrogels.
These results indicate chondrogenic differentiation
capacity of ASC, which can clearly be increased by contact
to gelatin type B-based hydrogels.
This work was financially supported by the Federal State
of Mecklenburg-Vorpommern, the research funding
FORUN of the Medical Faculty, University Rostock and the
Research Foundation - Flanders.
Keywords. adipose tissue-derived stem cells, gelatinbased hydrogel, chondrogenic differentiation, tissue
engineering
(1.P7) COMPARATIVE CHONDROGENIC PROFILE OF
RABBIT AND HUMAN ADIPOSE MESSENCHYMAL STEM
CELLS
Nicolàs M (1), Herrero-Méndez A (2), Castro B (2),
Fernández AG (1), del Olmo M (2), Guglietta A (1)
1. Ferrer International; 2.Histocell
Introduction. Articular cartilage injuries compromise the
quality of life for more than 30 million people per year.
Articular cartilage is unable to initiate a spontaneous and
efficient repair response when injured. Nowadays, there
are three strategies in regenerative medicine for cartilage
repair: implantation of chondrocytes (ACI), chondrocytes
seeded in a matrix (MACI) or the application of threedimensional hydrogels containing cells. These strategies
use almost exclusively chondrocytes, but in most cases
results are not very encouraging because: 1) new
cartilage formed is mostly fibrocartilage; and 2) obtaining
large amounts of autologous chondrocytes is extremely
difficult. The use of mesenchymal stem cells (MSCs) is a
promising alternative. To create a new product for
articular cartilage regeneration based on predifferentiated adipose MSCs (AMSCs), it is very important
to know the chondrogenic process in the human and in
the animal specie selected for the in vivo studies. Here we
compare the chondrogenic profile from human and rabbit
AMSCs. Objectives: To ascertain that the population of
cells from rabbit adipose tissue are MSCs and compare
the differentiation stage of hAMSCs with rabbit AMSCs
(rAMSCs). To define the optimal time for the
differentiation of human AMSCs (hAMSCs) in which these
cells not only present the characteristics of the chondral
linage, but also mantain their proliferative capacity.
Results. Cells obtained from rabbit adipose tissue are
MSCs, as they could be differentiated to bone, cartilage
and adipose tissue. Both, hAMSCs and rAMSCs show
similar phenotypic and genotypic characteristics of the
chondral linage at day four of differentiation. Moreover,
in this stage AMSCs are able to proliferate and form
colonies.
Conclusion. An optimal expression of chondrogenic
markers is obtained after 4 days of differentiation of
hAMSCs. Furthermore, in this stage rAMSCs show similar
characteristics as hAMSCs, making rabbit a good species
to evaluate the effectiveness of the product.
Keywords. Adipose stem cells, rabbit, cartilage
regeneration
(1.P8) A COMPARISON BETWEEN HUMAN AND SHEEP
ADIPOSE MESENCHYMAL STEM CELLS’ PROFILE
Castro B (1), Martínez D (1), Rodríguez C(2), Béjar J (3),
Lagunas C (4)
1. Histocell; 2. Urquijo Clinic; 3. San Juan de Dios Hospital;
4. Salvat Biotech
Introduction. Pseudarthrosis or non-unions are severe
complications in orthopaedic trauma care and occur in
10% of all fractures. Non-union is a pathological process
that happens when the fractured ends of a bone are
covered by fibrocartilage and therefore bone
consolidation never occurs. The optimal approach is to
combine an osteoinductor (cells or growth factors), with
an osteoconductor (bone graft substitute). In this sense,
we are developing a new autologous product which
combines mesenchymal stem cells from adipose tissue
(hASC), easily obtained from the patient by liposuction,
with a bone graft substitute. The potency of hASC for
bone regeneration is well established. However, to
progress towards human clinical trials, in vivo
experiments are required, being sheep a convenient
large-animal model. Hence, the aim of this work is to
ascertain that the obtained cells from sheep’s adipose
tissue are ASC and to draw a comparison of the
differentiation potentials and stages between hASC and
sheep ASC (sASC).
Materials and Methods. Cells are extracted from adipose
tissue of sheep, by surgical procedures, and human
volunteers by means of lipoaspiration. The obtained cells
populations of both species are assayed for: cell-doubling
time, colony forming units’ potential (CFUs),
immunophenotypical characterization by flow cytometry,
and differentiation potential to bone, cartilage and
adipose tissue.
Results. Cells obtained from sheep adipose tissue are
ASC, since they have the potential to differentiate into
the three mesodermal lineages: bone, cartilage and
adipose tissue. Despite the phenotypic similarities
between them, the higher proliferative rate that sASC
present appears to accelerate the differentiation
processes.
Conclusion. The cells obtained from subcutaneous
adipose tissue can be considered sASC. Therefore, they
could be a good model for efficacy phases of advanced
therapy medicinal products containing ASC. However, the
differences in proliferative rate and differentiation
potential in relation to hASC must be taking into account.
Keywords. Sheep, stem cells, bone regeneration
(1.P9) AUTOLOGOUS ADIPOSE MESENCHYMAL CELLS IN
A CAVITARY MANDIBLE DEFECT PROMOTE BONE
REPAIR.
Trejo CG (1), Manso FJ (2), Martín-López J (1), Gimeno MJ
(1), Gómez-Gil V (3), García-Honduvilla N (1)
1. Department of Medical Specialities, University of
Alcalá, Networking Research Center on Bioengineering,
Biomaterials and Nanomedicine (CIBER-BBN); 2. European
Technology Institute of Dental Sciences University of
Alcalá; 3. Department of Pharmacology, University of
Alcalá Networking Research Center on Bioengineering,
Biomaterials and Nanomedicine (CIBER-BBN)
Introduction. The mesenchymal stem cells from
subcutaneous adipose tissue (MSCat) are an
undifferentiated population which can be easily isolated
and expanded in vitro. The MSCat have high potential to
differentiate to other lineages. Our objective was
demonstrated that MSCat implanted in a defect in rat
mandible can be improving the regeneration bone.
Materials and Methods. MSCat were obtained from
subcutaneous adipose tissue (N=16) to apply in cell
therapy in the Wistar rat model of mandible repair. 106
autologous MSCat fluorchrome PK26 markers were
implanted. After 7, 14, 21 and 30 days the bone
reparation
were
studied
in
histological,
immunohistochemical (RunX2, Osteocalcin(OC), TRAP and
ED-1) and radiologic analysis.
Results. In a control group the bone reparation was a
deficient in all groups of study, reaching 15% to 30 days.
In MSCat group the defect was filled in 30% to 30 days
this regeneration was evident with a PKH26 marker
fluorchrome in repair site. The RunX2 expression was
observed maintenance in all times in MSCat group. OC
expression was observed only in MSCat group and
increase over time. TRAP and ED-1 only was expressed
around defect at 7 days in MSCat group.
Conclusion. Mandible defects are susceptible to improve
after cell therapy. After mesenchymal cells implantation,
the bone regeneration was two folder in the treated vs.
control group.
This work has been supported by CAM (S0505/MAT/0324) and CAM (S-2009/MAT/1472)
Keywords. Cell therapy, Mesenchymal stem cells from
adipose tissue, osteogenic regeneration and mandible
repair.
(1.P10)
HUMAN
MESENCHYMAL
STEM
CELLS
CHEMOTAXIS ASSAY UNDER A GRADIENT OF
INFLAMMATORY CYTOKINES
Royo-Cañas, M (1), Desportes P (1), Sanz-Moreno, J (2),
Alegre-Aguarón, E (3), García-Alvarez, F (4), MartínezLorenzo, MJ (5), Larrad, L (6)
1. Instituto Aragonés de Ciencias de la Salud. 2.
Universidad de Zaragoza; 3.Columbia University, NY; 4.
Servicio de Traumatología Hospital Clínico Universitario
Lozano Blesa; 5. Banco de Sangre y Tejidos de Aragón; 6.
Servicio de Inmunología Hospital Clínico Universitario
Lozano Blesa
Introduction. The ability of mesenchymal stem cells
(MSCs) to repair tissue damage is related to antiapoptotic
and trophic effects mediated by MSC-derived soluble
factors. MSCs exert immunosuppressive activities by
suppressing T- and B-cell proliferation, dampening the
generation of mature myeloid dendritic cells, and
inhibiting the proliferation, cytokine production and
cytotoxic activity of natural killer cells. MSCs express
various chemokine receptors supporting chemokine
induced migration and display the ability to preferentially
home to sites of anatomic lesions. Their
immunomodulatory properties, together withtheir tissuetrophic properties, make MSCs good candidates to treat
autoimmune diseases, like reumatoid arthritis. Different
preclinical models of autoimmune diseases clearly
demonstrate the beneficial effects of MSCs on injured
tissues by inhibiting immune inflammation and promoting
tissue repair.
Materials and Methods. In this study, we have performed
a chemotaxis assay adapted for adherent cells, using the
Ibidi system “μ-Slide Chemotaxis” in colaboration with
the Microscopy and Image Unit of the Instituto Aragonés
de Ciencias de la Salud. We have used the
Multidimensional Microscopy System with Real Time
Control Leica AF6000 XL, which made possible to study in
vitro and directly the response and migration of the MSCs
under the effect of different cytokines. Two large volume
reservoirs are connected by a thin slit, where the MSCs
are seeded. The reservoirs contain different
chemoattractant concentrations, generating by diffusion
a linear and stable concentration profile through the
connecting slit. The data have been analyzed with the
internet image analysis software Image J, complemented
with a migration plugging. We have studied the behaviour
of human MSCs isolated from tissue samples collected
during surgical operations, which were cultured under a
gradient of several cytokines involucrated in
inflammatory processes: IL-1β, IL-6 and TNF-α.
Results and Conclusion. We have observed slight
differences in the behaviour of the MSCs under the
different cytokine gradients, which could help to explain
MSCs immunomodulatory properties.
Keywords. mesenchymal stem cells, immunomodulation,
inflammation
(1.P11) STUDY OF THE IN VITRO CYTOKINE SECRETION
PATTERN OF HUMAN MESENCHYMAL STEM CELLS
DERIVED FROM DIFFERENT TISSUES
Alegre-Aguarón E (1), Desportes P (2), García-Alvarez F
(3), Castiella T (4), Larrad L (5), Martínez-Lorenzo MJ (6)
1. Columbia University, NY; 2. Instituto Aragonés de
Ciencias de la Salud; 3. Servicio Traumatología Hospital
Clínico Universitario Lozano Blesa; 4. Servicio Anatomía
Patológica Hospital Clínico Universitario Lozano Blesa; 5.
Servicio Inmunología Hospital Clínico Universitario Lozano
Blesa; 6. Banco de Sangre y Tejidos de Aragón
Introduction. Cartilage is among the tissues with the
highest prevalence of aging-associated pathologies, in
part due to the fact that adult articular cartilage has
limited regenerative and reparative capacities. Different
strategies have been drawn up to deal with this problem,
and amongst them the use of mesenchymal stem cells
(MSCs) stands out as a good alternative. MSCs are
multipotent cells capable of differentiate into several
mesoderm lineages, cartilage among them. They have
been isolated from different tissues such as bone
marrow, adult peripheral blood, umbilical cord blood,
synovial liquid and adipose tissue. Besides, MSCs are
immuneprivileged and display immunomodulatory
capacities, which make MSCs good candidates to treat
autoimmune diseases, like reumatoid arthritis. They are
also capable of secreting several bioactive molecules,
which include cytokines and growth factors. The
expression of some adhesion molecules could be
important to explain their homing capacity in different
organs. The aim of this study was to measure the cytokine
concentration in the supernatants of human MSCs
cultures that had been derived from different tissues, and
to asses whether there was any difference that could be
due to their different tissue of origin.
Materials and Methods. Tissue samples were collected
from 18 human patients during surgical operations. The
samples corresponded to knee Hoffa’s fat, subcutaneous
fat from hip or knee, bone marrow and synovial liquid.
MSCs were harvested by mechanic and enzymatic
digestion, and separated by centrifugation. They were
cultured in an expansion medium at 37°C under a 5% CO2
humid atmosphere. The measurement of the different
cytokine levels was performed with the xMAP®
technology of Luminex® Corporation, using the Milliplex™
MAP kit (Millipore).
Results and Conclusion. Human MSCs, independently of
their tissue of origin, secrete mainly IL-6, a proinflammatory cytokine, although their secretion does not
inhibite the in vitro differentiation of these cells.
Keywords. human mesenchymal stem cells, chondrogenic
differentiation, cartilage
(1.P12) IMMUNOMODULATORY PROPERTIES OF HUMAN
MESENCHYMAL STEM CELLS ISOLATED FROM DIFFERENT
TISSUES
Desportes P (1), Alegre-Aguarón E (2), Sanz-Moreno J (3),
García-Álvarez F (4), Martínez-Lorenzo MJ (6), Larrad L (5)
1. Instituto Aragonés de Ciencias de la Salud; 2. Columbia
University, NY; 3. Universidad de Zaragoza; 4. Servicio de
Traumatología. Hospital Clínico Universitario Lozano
Blesa; 5. Servicio de Inmunología. Hospital Clínico
Universitario Lozano Blesa; 6. Banco de Sangre y de
Tejidos de Aragón
Introduction. Mesenchymal stem cells (MSCs), or more
accurately multipotent mesenchymal stromal cells, are
multipotent cells capable of differentiating into several
mesoderm lineages, (bone, cartilage, muscle, adipose
tissue), and even of transdifferentiating into ectoderm
(neurons) and endoderm (lung epitelium) lineages. They
represent a useful model in the clinical approaches to a
great number of diseases, both in regenerative therapy
and in gene therapy. Beside these features, MSCs are
immuneprivileged and display immunomodulatory
capacities, which together with their tissue-trophic
properties, make MSCs good candidates to treat
autoimmune disorders. Our group has focused in the
study of osteomuscular diseases, reumatoid arthritis
among them, for more than nine years.The aim of this
study was to evaluate the immunomodulatory capacity of
human MSCs harvested from five different tissues: bone
marrow, adipose tissue from two different anatomic
locations (sucutaneous and intraarticular), synovial liquid
and cartilage, and to asses whether the diverse tissue of
origin
was
of
any
significance
for
their
immunomodulatory
properties.
We
have
also
investigated the immunomodulatory effects of MSCs both
on freshly activated lymphocytes and on long-term
activated ones.
Materials and Methods. The different tissue samples
were collected from 6 human patients during surgical
operations. MSCs were harvested by mechanic and
enzymatic digestion, and separated by centrifugation.
They were cultured in an expansion medium at 37ºC
under a 5% CO2 humid atmosphere. PBMCs were isolated
from blood samples from healthy individuals by density
sedimentation on Ficoll-Histopaque gradients. The
cocultures were maintained for 4-5 days in 6-well, 12-well
or 96-well transwell plates. Lymphocyte proliferation was
measured by either MTT assay or flow cytometry (5,6carboxyfluorescein diacetate succinimidyl ester (CFSE)staining).
Results and Conclusion. MSCs differentialy suppressed
human PHA-activated-T-cell proliferation, and this
inhibition was dependent on the time T-cells were
maintained activated before the coculture and on the
presence or not in the culture medium of IL-2.
Keywords. mesenchymal stem cells, immunomodulation,
chondrogenic differentiation
(1.P13) RAT MODEL FOR ADIPOSE DERIVED STEM CELLS
THERAPY
Colaco B (1), Oliveira P (2), Afonso P (1), Pires MJ (2),
Cabrita AS (3), Villar JM (4), Villar V (4)
1 .CECAV. Departamento de Zootecnia. Universidade de
Trás-os-Montes e Alto Douro, Portugal; 2. CECAV.
Departamento de Ciências Veterinárias. Universidade de
Trás-os-Montes e Alto Douro, Portugal; 3. CIMAGO, Univ.
Coimbra, Coimbra, Portugal; 4. Instituto de Biomedicina,
Facultad de Veterinaria. Campus Vegazana. Universidad
de León, España.
To avoid transplant rejections from an individual to
another, the new tissue engineering field is developing
techniques and biological substitutes that re-establish,
maintain or improve the damaged tissue function. To
achieve this goal, cells, three dimensional biocompatible
scaffolds and tissue inductor substances has been
investigated to produce the desired tissue in vivo.
Adipose derived stem cells (ADSC) have gained in the last
years a special relevance in the tissue engineering field
because of their plasticity properties.
The aim of this work was to study the behaviour of rat
multipotent cells from abdominal and inguinal adipose
tissue in different culture conditions. To assess the
changes occurred in culture we used optical microscopy,
electronic microscopy and flow cytometry.
We observed that cells obtained from rat abdominal and
inguinal fat expressed stem cell markers (CD29 and
CD73), and had the potential to differentiate in
adipocytes, chondrocytes and neurons. The ADSC
adhered well to agar, collagen type I and poliglycolic acid,
revealing potential for its future use in tissue engineering.
We conclude that rat is a good animal model for assessing
the adipose derived stem cells potential to differentiate
and form tissues in vivo.
(1.P14) AGE-ASSOCIATED IMPAIRMENT OF HUMAN
ADIPOSE-DERIVED
MESENCHYMAL
STEM
CELLS
ANGIOGENIC PROPERTIES
Efimenko AYu (1), Dzhoyashvili NA (1), Starostina EE (1),
Kalinina NI (1), Parfyonova YeV (1)
1. Lomonosov Moscow State University
Introduction. Tissue regeneration is impaired in aged
individuals. Adipose-derived mesenchymal stem cells
(ADSCs) are promising source for cell therapy. ADSCs
secrete many angiogenic factors and improve
vascularization of ischemic tissues. However therapeutic
benefit of autologous ADSCs from aged patients could be
modest, because of their impaired functions. Here we
analysed how donor age affects angiogenic properties of
ADSCs.
Materials and Methods. ADSCs were isolated from
subcutaneous and pericardial fat obtained from 30
patients during the coronary artery bypass surgery and
cultured for 2-3 passages. Expression and secretion of
angiogenic factors were measured as well as ability of
ADSCs conditioned media to stimulate tube formation by
endothelial cell.
Results. ADSCs from “young” (mean age 46,6±3,3 years,
n=7) and “elderly” (63,8±7,0 years, n=23) individuals had
CD90+/CD73+/CD105+/CD45-/CD31- immunophenotype
and percentage of these cells was similar in both groups.
mRNA levels of VEGF and PlGF were lower and content of
HGF mRNA was higher in cells from elderly patients. In
contrast to mRNA, VEGF level was 2,7-fold higher in
conditioned media of ADSCs from aged donors. HGF level
didn’t differ between age groups. Total tube length
formed by EA.hy926 cells in the presence of ADSCs
conditioned media inversely correlated with donor age (r
= -0,64, p=0,008). Blocking of VEGF by neutralizing
antibodies inhibited tube formation up to 50%.
Conclusion. ADSC angiogenic properties decline with
donor age. This at least partially explains why autologous
ADSC from aged patients have an impaired therapeutic
potential.
The study was supported by Russian Federal Agency of
Science and Innovation (grant #02.527.11.0007).
Keywords. adipose-derived mesenchymal stromal cells,
aging, angiogenesis
(1.P15) CALCIUM PHOSPHATES BONE SUBSTITUTES
PROMOTED DIFFERENT OSTEOGENIC DIFFERENTIATION
PROFILE OF HUMAN ADIPOSE DERIVED STEM CELLS
Müller C (1), Castellarnau C (2), Reina M (1)
1. Celltec-UB, Department of Cellular Biology, University
of Barcelona, Spain; 2. ADVANCELL Advanced in vitro cell
technologies SA, Barcelona Science Park, Spain.
Introduction. Human adipose derived stem cells and
biomaterials are two fundamentals key in bone tissue
engineering and regenerative medicine. This study aims
to
compare
biocompatibility
and
osteogenic
differentiation of human adipose derived stem cells
(ADSCs) seeded on different calcium phosphates bone
substitutes.
Materials and Methods. 1x105 ADSCs cultured with
proliferative (PM) or osteogenic medium (OM) were
seeded on 0.5g of Bio-Oss® (Geistlich, Switzerland), Bone
Ceramic® (Strauman, Switzerland), Cerasorb® (Curasan,
Germany), or KeraOss (Keramat, Spain) granules with
protein coating. Cell adhesion and viability were detected
by Alamar blue assay at 0,7,14 and 21 days. Cell
morphology was observed by SEM. Osteogenic
differentiation was evaluated by ALP assay kit and Realtime PCR to quantify gene expression of alkaline
phosphatase (ALP), osteonectin (ON) and osteocalcin
(OC).
Results. the highest percentage of adherent ADSC (76%)
was found in KeraOss with protein coating and lowest
(45%) in Bio-Oss. Cell´s number attached to the different
scaffolds increased on the time for PM and OM,
meanwhile this response was not expressed on BioOss.
Different grown profiles were showed for each kind of
scaffolds. These results were confirmed by SEM. The
results revealed KeraOss granules are able to induce the
highest ALP activity of all. On the other hand the Real
Time PCR assays showed overexpression of ALP and OC at
14 days on Bone Ceramic and Cerasorb. In addition at 21d
ALP, OC and ON were upregulated on KeraOss.
Conclusion. ADSC can attach and grown on all kinds of
bone substitutes with exception of Bone Ceramic. These
scaffolds induce the osteoblastic differentiation of ADSC
in PM however is more intense in OM. This effect
increase with protein coating. Depending on the material,
the ALP activity and expression level of osteogenic
markers changed. These results suggest that the greatest
biocompatibility and differentiation is presented on
KeraOss and Bone Ceramic granules.
Keywords. ADSC, scaffolds, osteogenic differentiation
(1.P16) SHOULD THE INFLUENCE OF PATHOLOGICAL
OBESITY BE CONSIDERED WHEN USING hASCs FOR
TISSUE ENGINEERING APPLICATIONS?
Stanco D (1), Arrigoni E (1), de Girolamo L (2), Salvatori L
(3), Niada S (1), Petrangeli E (3), Brini AT (1)
1. Department of Medical Pharmacology, School of
Medicine, Università degli Studi di Milano, Milan, Italy; 2.
IRCCS Galeazzi Orthopaedic Institute, Milan, Italy; 3.
Experimental Medicine and Pathology Department,
Università di Roma “La Sapienza”, Rome, Italy
Introduction. Some pathological condition, like obesity,
may influence the features of human adipose-derived
mesenchymal stem cells (hASCs). Indeed, adipose tissue
of obese patients shows a reduced pressure of oxygen,
involved in the up-regulation of pro-inflammatory genes
that could affect the properties of these cells.
Materials and Methods. We have isolated hASCs from
subcutaneous adipose tissue of normal-weight donors
(nS-hASCs, n=5, mean age 33±6 years, BMI=24±2) and
from pathological obese donors (ObS-hASCs, n=5, mean
age 43±10 years, mean BMI=43±5). We have also
collected visceral adipose tissue from the obese patients
(ObV-hASCs, n=5) in order to evaluate possible
differences in the expression of the cell inflammatory
phenotype. We have characterized hASCs clonogenicity,
immunophenotype and osteogenic potential. We have
also evaluated the effects of hypoxic treatment on obese
hASCs cells.
Results. The clonogenic potential of cell populations, is
strongly lower in ObS-hASCs than in normal-weight
patients (-51%), and it is greater in subcutaneous than in
omental tissue among obese patients (+142%). ObShASCs show a significantly higher doubling time in
comparison to nS-hASCs (+40%); moreover ObV-hASCs
doubling time is higher than its corresponding
subcutaneous cells (+29%). From the immunophenotipic
point of view, the expression of CD54, CD90 and CD166 is
significantly reduced in ObS-hASCs respect to normalweight patients. The osteogenic potential of hASCs is also
affected by obesity: indeed, a significant reduction in the
alkaline phosphatase activity and calcified extracellular
matrix deposition was observed. Preliminary data suggest
that both ObS- and OBV-hASCs are responsive to hypoxic
treatment resulting in the activation of pro-inflammatory
genes.
Conclusion. The pathological obesity negatively affects
the self-maintaining and differentiation ability of hASCs,
probably due to the inflammatory state related to this
conditions. Our data suggest that some pathological
condition should be considered before proposing the use
of hASCs in tissue engineering applications.
Keywords. adipose stem cells; pathological obesity;
differentiative ability
(1.P17) STUDY OF PORCINE ADIPOSE-DERIVED STEM
CELLS FOR TISSUE ENGINEERING
Arrigoni E (1), Stanco D (1), Sangalli F (1), Niada S (1), de
Girolamo L (2), Yenagi V (1), Brini AT (1)
1. Department of Medical Pharmacology, School of
Medicine, Università degli Studi di Milano, Milan, Italy; 2.
IRCCS Galeazzi Orthopaedic Institute, Milan, Italy
Introduction. Since adipose-derived stem cells (ASCs) may
represent a promising approach for osteochondral
defects treatment, we have characterized the osteogenic
and chondrogenic potential of pASCs (pig-ASCs) in
comparison to hASCs (human-ASCs).
Materials and Methods. We have isolated ASCs from
caudal pig adipose tissue and from patients undergone
aesthetic liposuction under informed consensus. We have
analyzed ASCs clonogenicity, proliferation, osteogenic
and chondrogenic potential. Moreover, we have also
evaluated the osteogenic ability of biocompatible
materials.
Results. pASCs proliferate faster than human cells with a
doubling time of 54h and 126h, respectively. ASCs of both
sources are highly clonogenic with about 15% of colonies
formation until the passage 4. On polystirene, both
osteogenic differentiated pASCs and hASCs show an
increased alkaline phosphatase (ALP) activity of about
100% respect to undifferentiated cells, even if the ALP
basal levels were 10-fold higher in hASCs respect to
pASCs. The presence of scaffolds seems to significantly
increase ALP level both for undifferentiated and
differentiated pASCs and hASCs. We have also observed a
synergistic effect produced by scaffold plus osteogenic
stimuli, supporting the future clinical applications of ASCs
bioconstructs.
Moreover,
both
chondrogenic
differentiated pASCs and hASCs, aggregated into
micromasses, express an abundant amount of GAGs
showing a significant increase in comparison to
undifferentiated cells.
Conclusion. We show that pASCs and hASCs share
common features and possess a similar differentiative
ability, supporting the idea that the pre-clinical
autologous ASCs reimplantation model in pig might be
predictive of the behaviour of ASCs in a future clinical
model of regenerative medicine.
Keywords. porcine adipose-derived stem cells,
biocompatible scaffols, osteogenic differentiation,
chondrogenic differentiation
(1.P18) HUMAN ADIPOSE DERIVED STEM CELLS RETAIN
THEIR CHONDROGENIC POTENTIAL DURING EXPANSION
WITH HUMAN PLATELET LYSATE
Hildner F (1), Wolbank S (2), Aberl J (1), van Griensven M
(2), Redl H (2), Gabriel C (1), Peterbauer A (1)
1. Red Cross Blood Transfusion Service of Upper Austria,
Linz, Austria; 2. Ludwig Boltzmann Institute for
Experimental and Clinical Traumatology, Vienna, Austria
Introduction. Fetal calf serum (FCS) bears a potential risk
for carrying diseases and eliciting immune reactions.
Nevertheless, it still represents the gold standard as
medium supplement in cell culture.
Materials and Methods. In the present study human
platelet lysate (hPL) has been tested as an alternative to
FCS for the expansion and subsequent chondrogenic
differentiation of human adipose derived stem cells
(ASC). ASC were isolated from liposuction material of 8
donors and expanded up to passage 3 with 10% FCS
(group 1) or 5% hPL (group 2). Subsequently, three
dimensional micromass pellets were created and cultured
for 5 weeks in chondrogenic differentiation medium
without hPL or FCS but supplemented with 10 ng/mL
bFGF and 10 ng/mL TGF-β3. In order to evaluate the
effect of hPL on chondrogenesis during cell condensation,
micromass pellets of group 2 were additionally treated
with 5% hPL within the initial 3 days of micromass pellet
culture (group 3).
Results. Growth curves revealed that medium
supplementation with hPL strongly increases cell
proliferation. Chondrogenic differentiation has been
evaluated by qRT-PCR, glycosaminoglycan (GAG)
quantification and histological staining. Ten cartilage
related markers (COL2A1, COL1A1, SOX9, COL9A2,
COL10A1, AGC1, CSPG2, MIA, COMP, CRTL1) were
evaluated with qRT-PCR and demonstrated chondrogenic
differentiation of both, hPL and FCS expanded ASC. GAG
quantification did not reveal significant differences
between the three groups, although hPL expanded cells
tended to express higher levels of GAG. Histologically,
collagen type II and GAGs could also be detected in all
groups.
Conclusion. The present study demonstrates that hPL
strongly induces proliferation of ASC while retaining the
chondrogenic differentiation potential, suggesting that
hPL is equal or superior to FCS as supplement for the
expansion of ASC particularly with regard to chondrogenic
differentiation.
The authors wish to express their thanks to Tamara
Jagersberger and Mag. Christa Hackl for excellent
technical assistance
Keywords. Human Platelet Lysate, PRP, Cartilage
(1.P19) HUMAN ADIPOSE EXTRACELLULAR MATRIX
SUPPORTS CHONDROGENIC DIFFERENTIATION OF
ADIPOSE MESENCHYMAL STEM CELLS
Ibsirlioglu T (1), Elcin AE (1,2), Elcin YM (1)
1. Ankara University, Stem Cell Institute, Faculty of
Science, TEBNL, Ankara, Turkey; 2. Gazi Univ., GEF,
Biology Div., Ankara, Turkey
Introduction. Adipose extracellular matrix (ECM) consists
of the secreted products of the resident cells of the
adipose tissue comprising a three-dimensional
ultrastructure and a unique composition that could be
useful for a variety of tissue engineering applications. The
purpose of this study was to evaluate decellularized
adipose tissue as a biologic scaffold for the
chondrogenesis of human adipose mesenchymal stem
cells (hAMSCs) and in-vitro cartilage-like tissue formation.
Materials and Methods. Adipose tissue of adult patients
were collected from elective operations under ethical
approval. A novel and practical protocol developed at our
laboratory was applied for decellularization. hAMSCs
were isolated from the adipose tissue, expanded and
characterized immunophenotypically and by their
differentiation potential. ECM and hAMSCs were
clusterized, cultured in 10% FBS containing DMEM-F12, at
37oC, 5% CO2 and 95% humidity. After a week, the
medium was switched to the chondrogenic medium and
cultured for 35 days under static and bioreactor
conditions. Cell viability, DNA content, formation of the
cartilage-like tissue were evaluated at regular intervals,
using MTT, picogreen assay, histology and IHC,
respectively.
Results. The biochemical and structural properties of the
adipose ECM can vary according to the selected protocol.
Here, adipose ECM was obtained in a reproducible way
and supported chondrogenic differentiation of hAMSCs.
Immunophenotypical characterization demonstrated
strong positivity for CD 90, CD73, CD105, CD29, CD166,
CD44, and was negative for CD34, CD45, CD133. The
clusterized ECM and the hAMSCs gained mechanically
stability over time, especially in course of the
chondrogenic culture process. The hAMSCs seeded inside
the ECM scaffold proliferated faster during the initial
culture period, and maintained in number during the
chondrogenic culture, confirmed by the MTT and
picogreen assays. Histology and IHC indicated the
formation of a cartilage-like tissue in-vitro.
Conclusions. Results point out the potential of
decellularized adipose tissue as a biologic scaffold for
cartilage tissue engineering.
Keywords. Adipose extracellular matrix, decellularization,
matrix technology, adipose mesenchymal stem cells,
chondrogenesis
(1.P20) 2D AND 3D MULTILINEAGE DIFFERENTIATION OF
HUMAN ADULT (ADIPOSE TISSUE DERIVED) AND
EMBRYONIC STEM CELLS FOR TISSUE ENGINEERING
Declercq H(1), T' Joen V (1), Cornelissen M (1)
1. Tissue Engineering Group, Department of Basic Medical
Science, Ghent University, De Pintelaan 185 (6B3), 9000
Ghent, Belgium
Introduction. Human adipose tissue derived stem cells
(AT-MSC) and embryonic stem cells (ESC) are promising
alternatives for mesenchymal stem cells (MSC). Abundant
amounts of adipose tissue can be obtained and these ATMSC have multipotent characteristics. ESC have unique
dividing capacities and pluripotent characteristics but
directed differentiation is necessary. In this study ATMSC, ESC-MSC and bone marrow (BM)-MSC are
compared on their: 1) expansion efficiency and
multilineage differentiation capacity.
Materials and Methods. AT-MSC (Cryosave) and BM-MSC
(Lonza) were expanded in MesenPRO. ESC-MSC were
derived from ESC (H1, VUB01) via different strategies.
Adipogenic, chondrogenic and osteogenic differentiation
was studied in 2D and 3D cultures. For osteogenic 3D
differentiation,
MSC
are
seeded
on
3D
scaffolds/microcarriers and cultured dynamically for 40
days. The cultures were evaluated by fluorescence
microscopy, histology and qRT-PCR.
Results. AT-MSC, BM-MSC and ESC have a population
doubling time of respectively 25, 54 and 36 hours. EB
formation of ESC is insufficient for large-scale
differentiation while predifferentiation of ESC in
monolayer culture resulted in a morphologically
homogeneous population of MSC-like cells. 2D adipogenic
differentiation was apparent by the accumulation of lipidrich vacuoles and was most obvious for AT-MSC followed
by BM-MSC and ESC-MSC. 3D chondrogenic
differentiation was achieved in pellet cultures, although
the extracellular matrix (ECM) stained less intense than
chondrocyte controls. AT-MSC had the highest osteogenic
differentiation capacity as demonstrated in 2D and 3D.
During 3D osteogenic differentiation on microcarriers, the
cell-seeded microcarriers formed clusters after 14-21
days. Histology of the scaffolds/microcarriers revealed
fully colonization and a bone-specific ECM formation
(figure 1). These data were confirmed with qRT-PCR.
Conclusions. AT-MSC have an excellent adipogenic and
osteogenic differentiation capacity in comparison with
ESC-MSC. ESC-MSC would offer an alternative source to
study cell/biomaterial interactions in vitro. However, the
predifferentiation of hESC to hESC-MSC should be
optimized to obtain a homogeneous population of MSC.
Keywords. human adipose derived stem cells, human
embryonic stem cells, tissue engineering, 3D culture
Figure 1. Osteocalcin immunostaining of colonized 3D
scaffolds with BM-MSC (a), AT-MSC (b) and ESC-MSC (c)
after dynamic culturing for 40 days in osteogenic
medium.
(1.P21) TRACHEAL RECONSTRUCTION BY MONOLAYERED
MESENCHYMAL STEM CELLS WITH SMALL INTESTINE
SUBMUCOSA IN A RABBIT MODEL
Kwon SK (1), Lim JY (2)
1. Dongguk University Ilsan Hospital; 2. Inha University
School of Medicine
Introduction. There are no proven methods of
construction of tracheal defects when end-to-end
anastomosis is considered impossible. Trachea
replacement using prosthetic or biological substitutes
have thus far yielded unsatisfactory results, preventing
their clinical application. The failures of these methods
have been mainly due to donor sites’ restriction,
immunologic complications, bacterial infections, and
material failure. We aim too investigate the tracheal
reconstruction by monolayered autologous mesenchymal
stem cells with small intestine submucosa in a rabbit
model.
Materials and Methods. Eleven male New Zealand white
rabbits were randomly divided into three groups: rabbits
with trachea defect without reconstruction (untreated
group, n=4), rabbits with trachea defect given small
intestine submucosa (SIS) graft (SIS group, n=4), and
rabbits with trachea defect that underwent
transplantation of monolayered mesenchymal stem cells
(MSCs) on SIS (SIS+MSC group, n=4). Histological and
endoscopic analyses were performed by hematoxylineosin staining (H&E), Prussian blue staining and
endoscopy.
Results. Morbidity and mortality in the SIS+MSC group
were minimal, compared to untreated group and SIS
group. The specimens obtained from untreated group
and SIS group showed severe infiltration of inflammation
cells and granulocytes into the tracheal lumen at 1 week
after operation. In the SIS+MSC group, however, minimal
infiltration of inflammation cells and granulocytes was
noted. Twelve weeks following the operation,
regeneration of pseudostratified squamous epithelium
and ciliated columnar epithelium were confirmed by H&E
staining with minimal inflammatory infiltration in
SIS+MSC group. Moreover, Prussian blue staining clearly
demonstrated the presence of labeled MSCs in the
regeneration tissue of SIS+MSC group.
Conclusion. Tracheal reconstruction by MSCs with SIS can
be used to reconstruct a rabbit tracheal defect with
minimal mortality and morbidity, which appears to be a
promising therapeutics in the treatment of patients with
tracheal defects.
Keywords. Trachea, reconstruction, cell sheet
(1.P22) THE THERAPEUTIC EFFECT OF ADIPOSE-DERIVED
STEM CELL AND BDNF-IMMOBILIZED SCAFFOLD IN A RAT
MODEL OF CAVERNOUS NERVE INJURY
Bae JH (1), Piao S (1), Kim IG (1), Lee JY (1), Oh SH (2), Lee
JH (2), Ra JC (3), Lee DS (4), Lee JY (1)
1. Department of Urology, College of Medicine, The
Catholic University of Korea, Seoul 137-701, Korea; 2.
Department of Advanced Materials, Hannam University,
Daejeon 305-811, Korea; 3. Stem Cell Research Center,
RNL Bio Co., Ltd. Seoul 153-768, Korea; 4. Yongsan
International School, Seoul 140-210, Korea
Introduction. Post-prostatectomy erectile dysfunction
(ED) is a serious side effect for prostate cancer patient,
and reduces the patient quality of life. In this study, we
investigated the effect of human adipose-derived stem
cells (h-ADSCs) and BDNF incorporated Poly-Lactic-CoGlycolic (PLGA) membrane combined therapy in a rat
model of bilateral cavernous nerve (BCN) injury.
Materials and Methods. Sprague-Dawley rats inflicted
with BCN crush-injury were used for animal model.
Experimental groups were divided 5 groups; normal (N),
BCN crush-injury (C), h-ADSC after BCN injury (A), BDNFPLGA membrane after BCN injury (B), and h-ADSC and
BDNF-PLGA membrane after BCN injury (AB). PKH26labeled h-ADSCs were applied around the injured
cavernous nerve, and then BDNF-released PLGA
membrane was immediately covered on. Four weeks
after operation, erectile function was assessed by
detecting intra-cavernous pressure (ICP). Cavernous
nerve and corpus cavernosum were collected for
histological and molecular examinations.
Results. We found that h-ADSCs engrafted into the
cavernous nerve under fluorescent microscopy. In
functional study, ICP in group C was decreased compared
with N, but ICPs in group A, B and AB were increased
compared with group C. In histologic examination,
collagen content in corpus cavernosum was increased in
group C, but a little changed group A, B and AB. Molecular
study showed that the application of h-ADSCs and/or
BDNF-PLGA membrane increased cGMP and eNOS
expression in corpus cavernosum after BCN injury. The hADSCs and/or BDNF-PLGA membrane combined therapy
was more effective than each single therapy.
Conclusion. These results suggested that application of hADSCs on bilateral cavernous nerve, covered with BDNFreleased PLGA membrane can prevent the corpus
cavernosum damage after BCN injury. So, this combined
approach may provide a novel therapy for postprostatectomy ED.
Keywords. Adipose-derived stem cell, Brain-derived
neurotrophic factor, Cavernous nerve, Erectile
dysfunction
(1.P23) REVERSIBLE IMMORTALIZATION OF HUMAN
ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS
Tátrai P (1), Szepesi Á (2), Szigeti A (2), Német K (2)
1. Department of Experimental Gene Therapy, National
Blood Transfusion Service, Budapest, Hungary; University
of Debrecen, Debrecen, Hungary: 2. Department of
Experimental Gene Therapy, National Blood Transfusion
Service, Budapest, Hungary; Creative Cell Ltd., Budapest,
Hungary
Introduction. Background and aims. Adipose tissuederived mesenchymal stem cells (adMSCs) can be easily
harvested from human donors and differentiated into the
standard osteogenic, chondrogenic and adipogenic
directions, as well as towards a putative endothelial
phenotype. However, heterogeneity between donors,
dependence of cellular properties on passage number,
and limited life span of in vitro adMSC cultures present
major hurdles for reproducible experiments. Therefore,
we aimed to establish immortalized adMSC populations
with well-characterized properties that can provide a
steady supply of homogeneous cells for in vitro work.
Materials and Methods. The immortalizing genes Bmi-1
and SV40 large T antigen, combined with hTERT, were
transduced using Cre-excisable lentiviral vectors into
adMSCs of a single donor. Transgene copy number was
determined by qPCR relative to RNase P. Expression of all
transgenes was verified by immunofluorescence and RTqPCR, and telomerase activity was measured using
TRAPeze assay. Cell surface markers were detected by
flow cytometry. Proliferation was assayed using resazurin
dye. Osteogenic differentiation was assessed based on
alkaline phosphatase immunodetection and enzymatic
activity, and sprouting assay for endothelial
differentiation was carried out in Matrigel.
Results. Both Bmi-1+hTERT and SV40T+hTERT cell
populations have preserved expression of MSC markers,
and both have been subcultured for over 30 passages
without any sign of senescence. However, the two
populations possess clearly distinct properties. While
Bmi-1+hTERT is a mixed population with morphology,
proliferation and differentiation comparable with the
parental adMSC, SV40T+hTERT has quickly become a
rapidly proliferating cell line.
Conclusions. Since Bmi-1+hTERT MSCs have maintained
close-to-native MSC features, they may be utilized
directly in differentiation experiments. SV40T+hTERT, on
the other hand, can be efficiently expanded, and may
possibly be reverted to a conservative MSC phenotype by
subsequent Cre-mediated removal of the immortalizing
transgenes. Financial support. This work was supported
by the grant TÁMOP-4.2.1 from the Hungarian National
Development Agency (NFÜ).
Keywords. adipose tissue-derived mesenchymal stem
cells, immortalization
(1.P24) ELECTROPORATION-MEDIATED TRANSFER OF
RUNX2 AND OSTERIX GENES TO ENHANCE
OSTEOGENESIS OF ADIPOSE STEM CELLS
Lee JS (1), Lee JM (1), Im GI (1)
1. Dongguk University Ilsan Hospital
Adult stem cells are the promising potential for
differentiation into several cell types and predominantly
the adipose stem cells (ASCs) obtained from lipoaspirates
has the multi-lineage prospective to differentiate into
various cell types. Several explorations have shown that
ASCs have the potential to differentiate into osteogenic
lineages by the transfection of BMP expression vectors.
The constraint for the use of BMP expression has low
efficiency of its expression in the exogenous in vivo
system during osteogenesis. To address these facts
several researchers have explored the use of alternative
bone specific transcription factors to induce efficient
osteogenesis. Transfection of Runx2 and osterix in
mesenchymal stem cells leads to the development of
osteoblastic cells and bone formation. However the
foremost negative aspect of viral transfection methods
are immunogenicity and mutagenesis for these reasons
much effort has been made to go for advantageous
nonviral transfection by electroporation method to
transfer the growth factor genes. In the present study, we
tested the hypothesis that electroporation-mediated
transfer of Runx2 and Osterix genes to provoke in vitro
and in vivo osteogenic potential in ASCs.
Acknowledgements. This work was supported by a grant
from the Korea Ministry of Education, Science and
Technology (Grant No 2010-0000305).
Keywords. gene transfer, ATMSCs, osteogenic
differentiation
2. BIOFABRICATION FOR
REGENERATIVE MEDICINE
APPLICATIONS
Chair: James J. Yoo
Co-chair: Wei Sun
Keynote speaker: James J. Yoo
Organizers: James J. Yoo, Wei Sun
Synopsis: Biofabrication has become an innovative tool
for tissue engineering and regenerative medicine.
Biofabrication
uses
cells,
biomaterials
and
macromolecules to create basic building blocks of tissues
and organs. This special session will report state-of-theart research and development of using novel physical,
chemical, biological, and/or engineering process for 1)
construction of cell assemblies as tissues for regenerative
medicine, disease models and drug models; 2) integrated
bio-nano fabrication and bio-micro fabrication; 3)
cell/tissue printing, patterning and organ printing; 4) cellintegrated biological systems, microfluidic devices,
biosensors, and biochips; 5) 3D tissue scaffolds and tissue
constructs; 6) Computer-aided biofabrication and tissue
engineering; and 7) Protein/biomolecule printing and
patterning.
(2.KP) BIOFABRICATION OF TISSUES FOR CLINICAL
TRANSLATION
Yoo JJ (1)
1. Wake Forest Institute for Regenerative Medicine
Advances in regenerative medicine have provided various
therapeutic opportunities in the field of medicine. While
tissue engineering and regenerative medicine have had
initial successes in building a number of tissues clinically,
challenges still exist in developing complex tissue
systems. One of the challenges that hamper rapid clinical
translation is due to the lack of efficient cell delivery
methods. Living tissues maintain inherent multi-cellular
heterogeneous structures, and rebuilding of such
complex tissue structures requires subtle arrangements
of different cell types and extracellular matrices at their
specific anatomical target sites. Biofabrication using an
inkjet printing technology has been proposed as a tool to
address this endeavor. In this session novel and versatile
methods of building tissue structures using biofabrication
technology will be discussed. Development strategies that
facilitate a rapid clinical translation will also be discussed.
Keywords. Biofabrication, Bioprinting, Translation
(2.O1) USE OF SILK FIBROIN AS A SUBSTRATUM FOR
HUMAN CORNEAL ENDOTHELIUM TRANSPLANTATION
Madden PW (1,2), George KA (1,3), Lai JNX (1,4),
Rodriguez G (1), Harkin DG (1,3), Chirila TV (1,2)
1. Queensland Eye Institute; 2. University of Queensland;
3. Queensland University of Technology; 4. University of
Melbourne
Introduction. Diseased or damaged corneas are surgically
removed and replaced with tissue from deceased donors.
Corneal transplantation could be improved by
engineering cell layer substitutes. This would overcome
the shortage of donors and improve quality. The
endothelial layer is vital to corneal clarity and with most
transplants this layer alone needs replacing. Our aim is to
grow an endothelial layer on a substratum suitable for
transplant.
Materials and Methods. Adult human corneal
endothelium has low proliferative activity. Most cells will
not mitose, but a small proportion can be stimulated to
divide by strong mitogens. We have used these along
with a free-floating sphere technique in preparing cells
with gross normal, ‘‘differentiated’’, morphology. To
function normally the endothelium is best introduced as a
confluent organised monolayer and to achieve this we
grew primary cells on silkworm (Bombyx mori) (BM)
fibroin, prepared as 5 μm thick membranes. Furthermore,
to try and improve cell attachment and growth without
the need for coatings, we investigated, 1) patterning the
fibroin surface and, 2) the use of an alternative fibroin
from Antheraea pernyi (AP) silkworms which contains the
RGD tripeptide site.
Results. Our BM fibroin membranes are transparent
(>96% transmission), strong, and should degrade
sufficiently slowly to allow the endothelial cells to
establish on the recipient cornea, yet still maintain
transparency to retain sight. We achieved cell confluence
with normal gross morphology. However, not only was a
collagen coating required, but also the membrane was
difficult to handle. To improve handling we manufactured
9mm diameter discs with a 1mm supporting ring. AP
membranes were difficult to prepare and required a
different method.
Conclusion. Silk fibroin can be prepared as a transparent
membrane that supports the growth of human corneal
endothelium with gross normal morphology. These
qualities further the potential application of fibroin for
clinical corneal endothelial transplantation.
Keywords. cornea: silk fibroin: endothelium
(2.O2)
MODULAR
TISSUE
FORMATION
WITH
CONFORMALLY COATED THERMO-RESPONSIVE RIGID
MICRO-TEMPLATES
Tekin H (1), Tsinman T (1), Ozaydin-Ince G (1), Gleason KK
(1), Demirel MC (2), Langer R (1), Khademhosseini A (3)
1. Massachusetts Institute of Technology; 2. Pennsylvania
State University; 3. Brigham and Women’s Hospital,
Harvard Medical School
Generating modular tissue units can be beneficial for
applications in tissue engineering, drug discovery, and
regenerative medicine. Recently, softlithographically
fabricated poly(N-isopropylacrylamide) (PNIPAAm) based
microstructures have shown promising results in the
release of cell aggregates, though the swelling of molds
during temperature changes may cause deformation on
cell clusters. In this study, biocompatible, elastic, and gas
permeable poly(dimethylsiloxane) (PDMS) was used to
fabricate rigid microstructures that were supplied with a
conformal coating of PNIPAAm using chemical vapor
deposition. At room temperature, conformal PNIPAAm
films on PDMS templates swelled to three times their
thickness at 37 °C. Combining both the stiffness and the
thermo-responsive properties of the resulting
microstructures, tissue constructs could be grown to
match the dimensions of the microgrooves and
furthermore easily retrieved at room temperature using
swelling property and hydrophilicity of PNIPAAm at 24 °C
(contact angle θ = 30° ± 2) compared to 37 °C (θ = 50° ±
1). Given these results, conformally PNIPAAm coated
PDMS microstructures can be integrated with traditional
microfabrication techniques and may become a versatile
tool for tissue engineering and drug discovery
applications.
Keywords. Thermo-responsive templates, modular
tissues, chemical vapor deposition, microfabrication
(2.O3) LASER-ASSISTED BIOPRINTING: A TECHNOLOGY
FOR DEALING WITH TISSUE COMPLEXITY
Guillemot (1), Kériquel (1), Guillotin (1), Souquet (1),
Catros (1), Fontaine (1), Bareille (1), Rémy (1), Fricain (1)
Amédée (1)
1. INSERM U1026; University of Bordeaux
Parallel to scaffold-based approaches, technological
advances in the fields of automation, miniaturization and
computer-aided design and machining have led to the
development of Bioprinting. This later concept has been
defined recently as the “the use of computer-aided
transfer processes for patterning and assembling living
and non-living materials with a prescribed 2D or 3D
organization in order to produce bio-engineered
structures
serving
in
regenerative
medicine,
pharmacokinetic and basic cell biology studies”. As
compared to traditional approaches in Tissue
Engineering, bioprinting represents a paradigm shift.
Indeed, its principle is not more to seed cells onto a
biodegradable scaffold but rather to organize the
individual elements of the tissue during its fabrication
step (before its maturation) through the layer-by-layer
deposit (bottom-up) of biologically relevant components.
Besides ink-jet printing and bioplotting by means of
pressure-operated mechanical extruders, the LaserAssisted Bioprinting (LAB) technology has emerged as an
alternative method, thereby overcoming some of the
limitations of ink-jet and micropen printing devices,
namely, the clogging (viscosity, cell agglomeration, ink
drying, etc...) of print heads or capillaries used by these
printers to achieve micron-scale resolution.
In this context, after describing physical parameters
involved in Laser-Assisted Bioprinting, we present its
applications for printing nanomaterials and cells, both in
vitro and in vivo and we discuss on how this highthroughput, high resolution technique may help in
reproducing local cell micro-environment, and hence
creating functional tissue engineered 3D constructs.
allowing to deal with tissue complexity and
heterogeneity.
Keywords. bioprinting, laser
(2.O4) FABRICATING SMALL DIAMETER, BRANCHED
VASCULAR SYSTEMS BY COMBINING INKJET PRINTING
AND MULTIPHOTON POLYMERIZATION
Kluger PJ (1,2), Borchers KA (1), Refle O (3), Engelhard S
(4), Meyer W (5), Novosel EC (2), Graf C (3), Bierwisch C
(6), Schuh C (1), Seiler N (4), Wegener M (5), Krüger H (5),
Jaeger R (6), Hirth T (1,2), Giller A (4) and Tovar GEM (1,2)
1. Fraunhofer Institute for Interfacial Engineering and
Biotechnology, Germany; 2. Institute for Interfacial
Engineering, University of Stuttgart, Germany; 3.
Fraunhofer Institute for Manufacturing Engineering and
Automation, Germany; 4. Fraunhofer Institute for Laser
Technology, Germany; 5. Fraunhofer Institute for Applied
Polymer Research, Germany; 6. Fraunhofer Institute for
Fraunhofer Institute for Mechanics of Materials, Germany
Introduction. To date only single in vitro engineered
tissues are transferred to clinical approaches due to
todays inability to fabricate suitable, artifical vascular
systems. Combining inkjet printing with high-resolution
multiphoton polymerization (MPP) enables us to generate
branched, tubular systems with diameters << 1 mm. New
synthetic polymers were tailored to match the needs of
the technical building process and the elastic properties
of blood vessels. The polymers were biofunctionalized to
achieve a close coating with endothelial cells (ECs).
Experimental Methods. Based on numerical simulations,
branched tubular scaffolds were fabricated by combining
inkjet printing and MPP. Precursor polymers, cross linking
agent, photo initiators and solvent additives were
optimized to yield photo reactive inks with customizd Emoduli. Crosslinked polymers were modified with
derivatized heparin and RGD and analyzed by XPS and
colorimetric
methods.
Viability,
proliferation,
functionality of primary human microvascular ECs on the
substrates was determined, using several assays and
immunocytological stainings.
Results. A set-up for integrating inkjet printing and MPP
has been designed with which branched vessel scaffolds
have been fabricated. The diameter of the tubes can
range between 20 µm and several millimeters (Figure 1).
Material compositions have been developed to achieve EModuli of 2-2000 MPa after crosslinking, the lower are
similar to natural blood vessels. Suitable after-treatment
ensured biocompatibility of the processed polymers,
thereafter thio-heparin and RGD have been covalently
bound on the surface. On these biofunctionalized
substrates an increased adhesion, viability and
proliferation of ECs has been determined in comparison
with unmodified substrates. EC-typical antigene
expression has been observed by immunocytological
stainings on all substrates.
Conclusion. The presented combination of rapid
prototyping techniques makes it possible to generate
small diameter vessel-like systems that can be applied for
supplying in vitro engineered tissues in a larger scale.
Acknowledgments. We thank the Fraunhofer Gesellschaft
for financial support to this project.
Keywords. artfical vessel scaffolds, inkjet printing and
multiphoton polymerisation, small diameter and
branched
(2.O5) NOVEL APPROACH TO AUTOMATING AND
SCALING UP PRODUCTION OF COLLAGEN BASED
SCAFFOLDS FOR THERAPY AND SCREENING
Drake RAL (1), Kaasi A (1), Purser MH (1), Brown RAB (2),
Cameron GWW (1)
1. The Automation Partnership; 2. University College
London
Introduction. Successful translation of research findings
into cost effective therapies requires process scale up for
production. Often manufacturing issues are overlooked
when materials and processes are being developed,
resulting in therapies that are difficult or expensive to
manufacture reproducibly. This is especially true for cell
therapies. Here we describe a new automated system for
production of biomimetic cell-containing collagen
scaffolds. Our aim is to enable reliable, consistent and
cost effective manufacture of cell-based therapies at a
commercial scale.
Materials and Methods. Brown et al (1) described a novel
approach to collagen engineering, plastic compression, in
which water is expelled from cell-seeded hyper hydrated
collagen gels. This simple technology allows direct
fabrication of strong, biomimetic tissues. Although the
manual process is rapid, taking less than 1 hour to make a
tissue, it is difficult to achieve good control of process
parameters. We have now developed a workstation to
automate and control the critical stages of the plastic
compression process.
Results. Data will be presented which show that the
manual process can be scaled up successfully. Multiple
tissues have been made in parallel in a variety of formats;
12, 24 and 96 multi-well plates. 3D cell seeded collagen
tissues with different cell types can be made rapidly and
reproducibly while retaining good cell viability. The
versatility of the system will be demonstrated by
reference to properties, such as multi-layering and
embossed surface features, that can be engineered into
tissues using this technology.
Conclusion. This workstation is an enabling platform
technology for making strong, collagen based tissues, and
a powerful tool for scientists developing novel tissues for
cell therapy or a wide range of research applications. It is
useful throughout the development process, supporting
process development, biomaterials development and
production of tissues for potency assays.
References 1. Brown R.A. et al. (2005) Adv. Funct. Mater.
15(11) 1762-1770
The work was supported in part by funding from the UK
Technology Strategy Board
Disclosures. TAP has licensed technology from UCL
Keywords. Translation; Cell therapy; Collagen scaffolds;
Scale up
(2.O6) DEVELOPMENT AND IN VITRO DEGRADATION OF
PLA/PEG/CaP GLASS BIODEGRADABLE SCAFFOLDS BY
RAPID PROTOTYPING
Serra T (1), Navarro M (1), Planell JA (2)
1. Institute for Bioengineering of Catalonia (IBEC); 2.
Institute for Bioengineering of Catalonia (IBEC); CIBERBBN; Technical University of Catalonia
Introduction. Rapid prototyping allows the development
of temporary 3D scaffolds with optimal architecture,
providing an adequate support for cell in-growth,
differentiation and ultimately tissue regeneration.
Particularly, a nozzle-deposition system integrated with
pumping technology is a versatile tool that uses a
CAD/CAM approach to build complex, reproducible 3D
structures. In this study, polylactic acid (PLA) and
polyethylene glycol (PEG) were combined with soluble
CaP glass particles and processed by RP to obtain fully
biodegradable structures with superior mechanical
properties and bioactivity. The aim of this work was the
development, characterization and in vitro degradation
study of biodegradable PLA/PEG and PLA/PEG/CaP glass
3D scaffolds.
Materials and Methods. A blend of 95%
Poly(95L/5DL)lactic-acid and 5% PEG (Mw=400) in
chloroform (5%w/v) was prepared. In the case of the
composite, CaP glass particles (<40um) in the system
44.5P2O5-44.5CaO-6Na2O-5TiO2 were also added (50%
w/w). Scaffolds with orthogonal and orthogonal-displaced
geometries were fabricated. The in vitro degradation
behaviour of the structures was evaluated by immersing
the scaffolds in SBF at 37°C for 8 weeks. Differential
scanning calorimetry, scanning electron microscopy
(SEM), mechanical compression test, micro-computed
tomography, and ionic (Ca2+) release were evaluated
after different degradation times. Biological evaluation
was also carried out.
Results. Well defined structures with 65% porosity were
obtained. Initial compression tests showed that both
geometry and glass particles affected the scaffolds
mechanical properties. Weight loss measurements and
SEM images (Fig.1) indicated that scaffolds were slowly
degraded loosing up to 7% of their initial weight and
increasing their surface microporosity. Nevertheless,
mechanical properties slightly decreased preserving the
scaffolds stability. Glass particles added an interesting
bioactive effect by releasing Ca to the medium. Indeed,
the addition of CaP-glass positively affected cell
behaviour.
Conclusion. The combination of RP and PLA/PEG/CaPglass
turned into promising fully degradable, mechanically
stable, bioactive and biocompatible composite scaffolds
for TE.
Keywords.
biofabrication,
rapid
prototyping,
biomaterials, biodegradable scaffolds, bone, regenerative
therapies
(2.O7) MICROWELL SCAFFOLDS FOR EXTRAHEPATIC
ISLET OF LANGERHANS TRANSPLANTATION IN TYPE 1
DIABETES
Buitinga M (1), de Koning EJP (2), Engelse MA (2),
Loomans CJM (2), Truckenmüller R (1), Moroni L (1), van
Blitterswijk CA (1), van Apeldoorn AA (1), Karperien M (1)
1. Department of Tissue Regeneration, University of
Twente, 7500 AE Enschede, the Netherlands; 2.
Department of Nephrology, University Medical Center
Leiden, 2333 ZA Leiden, the Netherlands
Introduction. The conventional therapy for type 1
diabetes is insulin administration. Despite this, some
patients are poorly controlled and suffer from
hypoglycemia and long-term complications. For these
patients, allogeneic islet transplantation into the liver has
become an alternative therapy[1]. Patients benefit from
this therapy due to near normalization of blood glucose
levels without an increased risk of hypoglycemia.
However, islet graft function in the liver tends to decline
over years indicating that the liver is not an optimal
transplantation site[2]. In order to develop alternative
transplantation sites with better long-term outcome, we
have developed a new microwell scaffold platform.
Materials and Methods. Microwell scaffolds were
prepared from dense solution-cast and porous
electrospun 4000PEOT30PBT70 block-copolymer films
using microthermoforming. Polymer wettability and
scaffold topology were assessed by captive bubble
contact angle measurements and scanning electron
microscopy (SEM), respectively. Furthermore, constructs
were characterized for their permeability for the nutrient
glucose. To determine the applicability of the constructs
for islet transplantation, the morphology and function of
human islets after 7 days of culturing were studied by
SEM, histological analysis and glucose challenge tests.
Results. We fabricated reproducible dense and porous
films, the latter with a fiber-diameter of 1.71±0.42µm.
The polymer films were hydrophilic (contact angle <40°) .
Diffusion tests revealed that the electrospun scaffolds
were permeable for glucose (flux: 0.0018±0.0002 gm-2s1). Based on SEM and histological analysis there were no
indications for islet spreading or outgrowth of islet
stromal cells. Function tests revealed that human islets
remained responsive to glucose challenge after 7 days of
culturing in the constructs (figure 1). Currently, first in
vivo trials are performed.
Conclusion. This study reports on the development of a
novel microwell scaffold platform for extrahepatic islet of
Langerhans transplantation. Alternative transplantation
sites using biomaterial scaffolds may improve islet
transplantation outcome.
[1]A.M.Shapiro et al.N Engl.J.Med,343,230-238(2000)
[2]E.A.Ryan et al.Diabetes,54,2060-2069(2005)
Keywords. Islet transplantation, Biomaterial, Scaffold,
Diabetes
(2.O8) MICROFLUIDICS FABRICATION OF SELFASSEMBLED
POLYSACCHARIDE
PEPTIDE
MICROCAPSULES FOR CELL THERAPY
Mendes AC (1), Baran ET (1), Reis RL (1), Azevedo HS (1)
1. 3B´s Research Group (Biomaterials, Biodegradables and
Biomimetics)
Self-assembling is an appealing methodology for the
bottom-up fabrication of new biomaterials that can be
used for the controlled growth of cell populations for cell
therapies or to promote regenerative processes in vivo.
Peptides are excellent structural units to form complex
nanostructures that can recreate some of the
architectural features of the natural extracellular matrix,
as they can self-assemble into fibril nanostructures. We
report here a mild cell encapsulation method based on
triggering the self-assembly of a multidomain peptide in
presence of xanthan gum polysaccharide, which has been
investigated in our group as artificial matrix for the
encapsulation of chondrocytic cell. The self-assembling
peptide K2(QL)6K2 has a central block of glutamineleucine (QL) repeats, and two flanking positively charged
lysine (K) to bind to the negatively charged xanthan. Using
a microfluidic device we were able to produce
microcapsules with homogenous size (diameter of 300
mm) by forming a water-in-oil multiphase. This
technology allows a control over the properties of the
microcapsules in terms of size and morphology, being a
low stress inducing method suited for cell encapsulation.
The properties and performance of xanthan-peptide
microcapsules
were
optimized
by
changing
peptide/polysaccharide ratio and their effects on the
microcapsules permeability and mechanical stability were
analyzed. Moreover, the effect of microcapsule
formulation on viability and proliferation of encapsulated
chondrogenic cells were also investigated. The
encapsulated ATDC5 cells were metabolically active,
showing an increased viability and proliferation over 21
days of in vitro culture demonstrating the long-term
stability of the developed microcapsules and their ability
to support and enhance the survival of encapsulated cells
over prolonged time. Combining self-assembling
materials with microfluidics processing proved to be
innovative approach to fabricate suitable matrices for cell
encapsulation and delivery.
ACM acknowledges to FCT for the financial support (PhD
grant SFRH/BD/42161/2007)
Keywords. Peptide self-assembly; Xanthan gum;
Microfluidics. Cell encapsulation, Microcapsules
(2.O9) FABRICATION OF A CUSTOMIZED TISSUE
ENGINEERING SCAFFOLD FOR BREAST RECONSTRUCTION
Wiggenhauser PS (1), Melchels FPW (2), Hutmacher DW
(2), Machens HG (1), Ong FR (3), Schantz JT (1)
1. Muenchen Rechts der Isar, Technische Universitaet
Muenchen; 2. Institute of Health and Biomedical
Innovation, Queensland University of Technology; 3.
School of Mechanical and Aeronautical Engineering,
Singapore Polytechnic
Introduction. Mastectomy can be necessary in breast
cancer therapy. To improve the patient’s quality of life,
plastic surgeons often reconstruct the breast. The stateof–the-art procedure is the transplantation of free fat
grafts from the belly to the breast. Disadvantages are
long operation times and risk of hematoma, infections or
donor site defects. A tissue engineered and vascularized
adipose construct could overcome these disadvantages
and could mimic the natural breast in respect of shape,
ptosis and touch. Tissue engineering scaffolds are needed
to shape the breast and support fat formation. Here we
demonstrate a method that is close to clinical reality,
using CAD/CAM technologies.
Materials and Methods. The body of a young female
patient is scanned with a 3D laser scanner from three
different angles. These scan images are digitally merged
and converted to a 3D model of the patient’s body. This
3D model is imported into CAD software. Software
algorithms are used to mirror the healthy breast and to
adapt this designed breast to the predicted thorax shape,
so that the scaffold fits to the recipient area of the
removed breast. Furthermore, CAD date are transferred
to rapid prototyping commands (STL language) and used
to fabricate a full-size breast scaffold with fused
deposition modeling.
Results and Conclusion. In conclusion, geometrically
complex scaffolds can be manufactured individually and
customized with 3D laser scanning, CAD modeling and
rapid prototyping.
Keywords. breast reconstruction, customization, clinical
setting, rapid prototyping, CAD, CAM
biomedical field. Under the maintenance of mechanical
properties and photoreactivity of conventional
photo¬polymerizable
monomers
based
on
(meth)acrylates, cytotoxicity and the degradation
behaviour could be significantly improved.
Keywords. Additive Manufacturing Technology
(2.O10) 3D-STRUCTURING OF POLY(VINYL ALCOHOL)BASED PHOTOPOLYMERS
Stampfl J (1), Schwentenwein M (1), Heller C (1), Varga F
(2), Russmüller G (3), Liska R (1)
1. TU Wien; 2. LBI Osteology; 3. Medical University of
Vienna
The fabrication of 3D-scaffolds with defined pore
geometries which enable good adhesion of cells is a
challenging topic in the field of regenerative medicine.
Photopolymers which can be structured by means of
Additive Manufacturing Technologies are promising
materials for this application. The possibility of
structuring these compounds via processes such as
microstereolithography (µSLA), Digital Light Processing
(DLP) or Two Photon Polymerization (2PP) enables the
fabrication of constructs with complex geometries and
high resolution mimicking cellular structures of natural
materials such as bone.
Beside the considerable irritancy and sometimes toxicity
of acrylate-based monomers, the formation of polyacrylic
acid through hydrolytic degradation of the polymer is
another undesirable aspect of these materials when
applied in the biomedical field. Therefore, photopolymers
with different polymerizable groups such as vinylesters,
vinylcarbonates and vinyl-carbamates which give watersoluble poly(vinyl alcohol) upon hydrolytic degradation,
were evaluated. Several monomers were synthesized to
examine the properties of these substance classes with
focus on cytotoxicity, photoreactivity, mechanical
properties and degradation behavior. 3D-parts made of
the new materials were implanted into New Zealand
White Rabbits to examine the behaviour under
physiological conditions. The biocompatibility of these
new substances, measured by their cytotoxicity towards
osteoblast-like cells, showed better results than for their
(meth)acrylate-based counterparts. The photoreactivity
was found to be between that of acrylates and
methacrylates, mechanical properties were on the same
level and degradation characteristics could be tailored
over a broad range. The in-vivo studies showed excellent
biocompatibility of the materials as well as
osteoconductivity due to the layered structure inherent
to parts structured with conventional AMTs.
The prepared photopolymers based on poly(vinyl alcohol)
show interesting properties for the application in the
(2.O11) BIOFABRICATION OF THREE-DIMENSIONAL
COMPLEX CONSTRUCTS VIA MAGNETIC DIRECTED
MICROGEL ASSEMBLY
Xu F (1), Rengarajan V (1), Finley TD (1), Sung Y (1),
Sridharan B (1), Demirci U (1)
1. Harvard Medical School
Introduction. Directed assembly of microgels is a
promising method for constructing complex threedimensional (3D) geometries that mimic native tissues.
Although several methods have been developed to
assemble the microgels, these methods are limited by
process complexity, low throughput potential, and the
use of organic solvents. A simple directed assembly
process with high throughput potential is still an
unsatisfied step towards recreating in vivo tissue
structures and functions. Here we propose a novel
magnetic directed assembly method for fabricating 3D
construct using microgels. Magnetic nanoparticles (MNPs)
were encapsulated in the microgels and manipulated
using externally applied magnets.
Materials and Methods. Microgels (PEG 1000) of
different sizes and shapes were fabricated using
photolithography. These microgels were fabricated by
encapsulating iron (II, III) oxide MNPs within the
hydrogels. These M-gels (magnetic nano-particle
encapsulated microgels) were attracted with the use of
neodymium magnets of 1 tesla power (Figure 1a). They
were secondary cross-linked along with 5 µl prepolymer
solution for the stability of the structure.
Results. Complex 3D constructs of the microgels were
achieved through magnetic directed assembly with
precise spatial control. We observed the formation of a
10 mm diameter single layer spheroid within 5 seconds of
introducing the magnetic rod. With such time control and
varying the concentration of MNPs (0.3%-2%) multi-layer
spheroids were fabricated (Figure 1b). We achieved a
various complex structures using the flexible templates
such as arc, dome, sphere and tubular constructs (Figure
1c-f). The observed M-gel assembly confirm that the
gravitational force can be balanced by the magnetic force
applied via permanent magnets.
Conclusion. Here we reported a directed assembly
method of microgels as building blocks via magnetics into
larger constructs that mimic in vivo structures. These
results envisage that this method holds the potential to
impact multiple fields including tissue engineering, stem
cell
technology,
regenerative
medicine,
and
pharmacology.
Keywords. Magnetics directed assembly, microgels,
complex constructs
(2.O12) TISSUE ENGINEERED CARTILAGE FROM HUMAN
BONE MARROW MESENCHYMAL STEM CELLS SEEDED IN
PLGA/SOX-TRIO GENE IN VITRO
Lee JS (1), Kim HJ (1), Im GI (1)
1. Dongguk International Hospital
Introduction. Articular hyaline cartilage injuries still pose
a big challenge to orthopaedic surgeons, because these
defects have poor capacity for instinsic repair. Tissue
engineered cartilage constructed with a combination
mesenchymal stem cells and three-dimensional
biomaterial may be a viable therapeutic option. The aim
of this study is to investigate the chondrogenic potential
of SOX-5, 6 , 9 as chondrogenesis related transcription
factors, using SOX-genes conjugated PLGA scaffold.
Methods. Five modifications of porous PLGA scaffolds
were tested: 1) PLGA/pEGFP-C1; 2) PLGA/SOX-5; 3)
PLGA/SOX-6; 4) PLGA/SOX-9; 5) PLGA/SOX-trio in terms of
cell proliferation and chondrogenic potential. Bone
marrow mesenchymal cells were seeded on PLGA
scaffolds, respectively. After three weeks, cells were
analyzed for DNA contents, GAG amount and real time
PCR. The rabbits were anesthetized, and the right legs
were prepared as described in the previous section. The
knee joint was exposed by medial parapatellar incision,
and the trochlear groove was exposed by lateral
dislocation of the patella. A 3 mm outer diameter
trephine drill was used to create osteochondral defects
(diameter 7 mm, thickness 2 mm) in the trochlear groove
of femur. The animals were divided into three groups: in
Group I, the defect was filled with PLGA scaffold ; in
Group II, the defect was filled PLGA scaffold seeded with
ASCs ; in Group III, the defect was filled SOX-trio
incorporated PLGA seeded with ASCs. To prepare the
implantation, ASCs (5 x 105 cell in 40µl) were suspended
in DMEM/F-12 media and injected inside the scaffold.
Five rabbits were allocated to each group. The patella was
repositioned and the capsule was repaired with 4-0 nylon
sutures. The rabbits were allowed to feed freely in their
cage immediately after the operation without a cast.
Rabbits were sacrificed after 8 weeks.
Results. When cultured in hASCs seeded in SOX-trio
plasmid incorporated scaffolds with chondrogenic
medium produced significantly richer ECM than did
control vector and single SOX-5,6,9 plasmid incorporated
scaffolds. Interestingly, real time PCR analysis
demonstrated that hASCs seeded in SOX-trio plasmid
incorporated scaffolds showed significantly higer gene
expression of type II collagen compared with control
vector and single SOX-5,6,9 plasmid incorporated
scaffolds .However, after 3 weeks in culture, there was
weak expression of type I and X collagen in SOX-trio
incorporated scaffolds group, but there was no
significantly difference in expression of genes in any other
groups. These findings indicate that SOX-trio incorporated
scaffolds a higher rate of chondrogenic potential than in
any other groups. In an in vivo osteochondral defect
model, treatment with PLGA scaffolds and SOX-trio
incorporated PLGA scaffolds demonstrated some ability
to potentiated cartilage regeneration. SOX-trio treatment
led to greater cartilage regeneration than in PLGA scaffols
only and ASC with PLGA scaffolds groups. At week 8,
macroscopic and histologic assessment demonstrated
that treatment with SOX-trio incorporated scaffolds
produced good articular cartilage healing with Safranin-O
positive hyaline cartilage.
Conclusion. In conclusion, our results suggest that SOXtrio incorporated plasmid DNA loaded scaffolds have
higher chondrogenic potential in vitro and induce more
cartilage regeneration in an in vivo osteochondral defect
model than do control groups. These results further the
understanding of the chondrogenic potential of SOX-trio
plasmid loaded scaffolds and may contribute to the
development of new therapeutic strategies for cartilage
repair and regeneration. Finally, we note that the gene
expression
is
occurring
within
the
scaffold
microenvironment, and that stimulus to promote
cartilage regeneration by the transgene expression must
be considered within the context of the
microenvironment, which
contains
architectural,
mechanical, chemical, and biological cues. All aspects of
the microenvironment created by the scaffold must be
considered for its role in promoting tissue formation, and
continued development of gene releasing scaffolds holds
great promise for numerous applications in regenerative
medicine.
Acknowledgement. This work was supported by a grant
from the Korea Ministry of Health Welfare (Grant No
A080061).
Keywords. plasmid incoporated scaffold
(2.O13) CELL PRINTING FOR 3D TISSUES
Zhang T (1), Zhang L (1), Zhang R (1), Lin F (1), Hamid Q
(2), Snyder J (2), Wang C (2), Sun W (1,2)
1. Department of Mechanical Engineering, Tsinghua
University, Beijing, P.R. China; 2. Department of
Mechanical Engineering, Drexel University, Philadelphia,
USA
In the new paradigm of tissue science and engineering,
living cells and biomolecules are used as basic building
blocks for biofabrication of cell-integrated medical
therapeutic products and/or non-medical biological
systems with applications found as tissue substitutes, 3D
cell and organ biological models, microfluidic biochips
and biosensors, and tissue models for study of disease
pathogenesis, drug discovery and toxicity testing. This
presentation will introduce our recent research in the
emerging field of cell printing and report our work on
using additive technology for direct cell writing for
construction of 3D cell assemble and tissue structures.
Presentation topic will include: 1) introduction of direct
cell writing process; 2) effect of the process parameters
on cell survivability; 3) characterization of biological
responses of various cells to the printing process; and 4)
applications to the field of tissue science and engineering.
(2.O14) INNOVATIVE THREE-DIMENSIONAL PLATFORM
FOR COMBINATORIAL ANALYSIS OF CELL/BIOMATERIALS
INTERACTIONS
Salgado CL (1), Oliveira MB(1), Mano JF (1)
1. 3B’s Research Group - University of Minho
Introduction. High Throughput (HT) systems are an
uprising area for analysis of biomaterials properties and
cell response to substrates. Combinatorial screening
allows for the selection of combinations of biomaterials
and/or bioactive agents in a preliminary stage of
physicochemical characterization or cell behavior
assessment. This leads to time and economically effective
studies. To have a closer approach to in vivo settings,
studies in three-dimensional (3D) conditions must be
performed.
An innovative top-down photolithographic approach is
here proposed to obtain biochip platforms for
material/cell interaction studies using biomimetic
polystyrene superhydrophobic surfaces (SHS). Cell
encapsulation in alginate-based hydrogels was used for
proof of concept.
Materials and Methods. The biochips were prepared by:
• Preparation of polystyrene SHS by a phase-inversion
method;
• Generation of superhydrophilic spots by exposure of
the SHS to UV/ozone radiation using hollow masks (Fig.
1A);
• Deposition of polymeric solutions mixed with cells,
further crosslinked with CaCl2.
Results and Discussion. Superhydrophilic spots with
controlled shape (squares) could be fabricated in the SHS.
Alginate-based hydrogels could be deposited in the spots,
keeping separate due to the wettability contrast between
the spots and the rest of the substrate, even after
immersion in cell culture medium.Two different cell lines
- osteoblast-like (MC3T3) and fibroblast-like (L929) –
previously encapsulated in the hydrogel matrices were
studied after 24 hours of cell culture. The composition of
the hydrogels affected cell response, leading to expected
tendencies for the well-known polymer mixtures (shown
in Fig. 1B).
The evaluation of cell viability and proliferation was
performed by direct methods (“chip-destructive”
Materials and Methods: MTS and DNA quantification) and
indirect methods (Calcein and DAPI staining image
analysis). The results of both methods were consistent
(Fig. 1B).
Conclusions. A biomimetic-inspired 3D biochip allowed
for HT cell culture study and result analysis of
combinatorial polymeric blends.
Acknowledgments. Mariana Oliveira acknowledges the
FCT PhD grant SFRH/BD/71396/2010.
Keywords. High-Throughput; Superhydrophobic Surfaces;
Biomaterials; Cell encapsulation
(2.O15)
ACOUSTICS
DIRECTED
MICROPARTICLE
ASSEMBLY FOR BIOMEDICAL APPLICATIONS
Xu F (1), Gurkan UA (1), Finley TD (1), Türkaydın M (1),
Yavuz AS (1), Demirci U (1)
1. Harvard Medical School
Introduction. Directed assembly of microgels holds great
potential for applications in tissue engineering and
regenerative medicine. However, there are several
limitations associated with the existing techniques
(hydrophilic-hydrophobic interactions, surface template)
such as complexity of assembly process, involvement of
organic solvents. There is still an unmet need for
straightforward assembly methods. Acoustic techniques
are emerging technologies offering several advantages
such as decreased instrumentation complexity and
gentler handling of pressure and heat sensitive biological
moieties such as cells. However, acoustics have not been
used for microgel assembly.
Materials and Methods. In this study we have developed
a novel acoustic assembler to assemble microgels, Figure
1a. Microgels (PEG 1000) of different shapes were
fabricated using photolithography. The microgels were
deposited onto the hydrophobic surface of a petri dish
where 40µL of deionized water was added to the group of
microgels. The petri dish was placed above a piezo buzzer
(Digi-Key, CPE-827) and exposed to acoustic vibrations
produced by a pulse/function generator.
Results. To evaluate particle manipulation with our
acoustic assembler, we assembled glass microbeads
(Figure 1b-c) and microgels with different shapes (Figure
1d-e). After applying acoustic excitation, the microbeads
came together at the center of the droplet within 30 sec,
Figure 1b. We observed that the microbeads assembly
time was dependent on excitation frequency, Figure 1c.
During acoustic excitation, we observed that some
microgels were immobile due to settling on their
untreated surface. It was determined that a frequency
sweep provoked mobility in the microgels more so than
using a constant frequency, leading to the assembly of
orientation specific microgels, Figure 1d-e.
Conclusions. In this study we report an acoustic
assembler that utilizes microscale hydrogels as building
blocks to create larger constructs via external acoustic
fields. This approach has potential to impact multiple
fields including tissue engineering, regenerative medicine,
and pharmacology.
Keywords. Microparticle assembly, acoustics, microgels
(2.O16) MANDIBULAR RECONSTRUCTION USING AN
AXIALLY
VASCULARIZED
TISSUE
ENGINEERED
CONSTRUCT
Eweida AM (1), Nabawi AS (1), Marei MK (2), Khalil MR
(1), Elhammady HA (1)
1. Department of Head and Neck and Endocrine Surgery,
Faculty of Medicine, University of Alexandria, Egypt; 2.
Tissue Engineering Laboratories, Faculty of Dentistry,
University of Alexandria, Egypt
Introduction. Tissue engineering and Regenerative
medicine depend mainly on the so-called extrinsic mode
of neovascularization, where the neovascular bed
originates from the periphery of the construct. This
method is not applicable for large defects in irradiated
fields.
Materials and methods. We are introducing a new animal
model for mandibular reconstruction using intrinsic axial
vascularization by the Arterio-Venous (AV) loop.
Cadaveric, mechanical loading, and surgical pilot studies
were performed on adult male goats. The cadaveric study
aimed at defining the best vascular axis to be used in
creating the AV loop in the mandibular region.
Mechanical loading studies (3 points bending test) were
done to put a base line for further mechanical testing
after bone regeneration. A pilot surgical study was done
to ensure smooth operative and post operative
procedures.
Results. The best vascular axis to reconstruct posterior
mandibular defects is the facial artery (average length
32.5±1.9mm, caliber 2.5mm), and facial vein (average
length 33.3±1.8mm, caliber 2.6mm). Defects in the
anterior half require an additional venous graft. The
designed defect significantly affected the mechanical
properties of the mandible (P value 0.0204). The animal
was able to feed on soft diet from the 3rd postoperative
day and returned to normal diet within a week. The
mandible did not break during the period of follow up (2
months).
Conclusions. Our model introduces the concept of axial
vascularization for mandibular reconstruction after
irradiation. This is the first study to introduce the concept
of axial vascularization using the AV loop for angiogenesis
in the mandibular region. Moreover, this is the first study
aiming at axial vascularization at the site of the defect
without any need for tissue transfer (in contrast to what
was done previously in prefabricated flaps). Qualitative
and quantitative data on angiogenesis and osteogenesis is
now being further studied by our team.
Keywords.
Mandibular
reconstruction,
Axial
vascularization, Bone regeneration, New animal model
(2.P1) ALGINATE FOAMS FOR TISSUE ENGINEERING
Andersen T (1), Melvik JE (1), Dornish M(1)
1. FMC BioPolymer AS, Industriveien 33, N-1337 Sandvika,
NORWAY
Scaffolds are important tools in the development of
applications within tissue engineering and regenerative
medicine. Scaffolds made from calcium cross-linked
alginate foams are both biocompatible and
biodegradable. This study presents alginate foams with
controllable physical characteristics. Alginate foams are
produced by mechanically agitating a dispersion of an
aqueous solution of alginate, plasticizers, foaming agent,
gelling agent and slowly hydrolyzing acid. An insoluble
gelling ion salt, e.g. CaCO3, is used and Ca2+ ions are
released as pH is lowered induced by the hydrolysis of Dglucono-delta-lactone. The wet alginate foam is then cast
in specific shape using a mold, kept at ambient conditions
to complete the gelling reaction, and then dried in an
oven at 35-80degC. The integrity of the foams was
measured using an SMS Texture Analyzer with tensile
grips after the dry foam was re-hydrated in a model
physiological solution. Both formulation and process were
modified to produce foams with different physical
characteristics as shown in the figure. Increasing the
particle size of CaCO3 from 4 to 20 µm resulted in
increased pore size and decreased foam strength.
Increased saturation levels of gelling ions from 25 to
125% led to decreased pore size and increased foam
strength and stability. There was a relationship between
foam strength and alginate molecular weight. However,
at similar molecular weights, stronger foams were formed
using a G-rich alginate than an M-rich alginate. Generally,
the foams have high pliability, they may be cut into
specific shapes and sizes, they are not sticky, they can
easily be folded and refolded after hydration, and they
can be sutured. The foam will easily dissolve by adding
agents that chelate Ca2+ such as citrate. Modifications of
alginate foam formulations and the production process
can be used to construct foams with physical and
functional properties tailored for tissue engineering
applications.
Keywords. Alginate, scaffold, tissue engineering
(2.P2) THE ADVANTAGE OF COLLAGEN COATING FOR
DIFFERENT BIODEGRADABLE MATERIALS USED IN
REGENERATION OF THE ABDOMINAL TISSUE
Antoniac IV (1), Albu M (2), Miculescu F (1), Antoniac A
(1), Cotrut C (1)
1. University Politehnica of Bucharest, Romania; 2.
Collagen Department, INCDTP – Division Leather and
Footwear Research Institute, Bucharest
Introduction. Collagen is proved to be a good biomaterial
for use as biomedical implantable device due to its weak
antigenecity, excellent biocompatibility, controlable
resorbability and ability to integrate with surrounding
tissues. It acts as a regenerative template and the
collagen which covered implant is progressively degraded
and replaced by new cell-synthesized tissue. For implants,
the formation of new connective tissue, particularly
collagen, plays a key role. The aim of this study was to
investigate
physical-chemical
and
morphological
characteristics of collagenated meshes and to evaluate in
vitro biodegradability and biocompatibility and host
tissue response to the prosthetic biomaterials.
Materials and Methods. Different reinforcement meshes
for abdominal surgery, made by polypropylene and
polyester were impregnated with type I collagen gel, 1.1%
(w/w) at 7.2 pH, plasticized with 2% (w/w) glycerin and
cross-linked with 0.2% (w/w) glutaraldehyde. Every mesh
was immersed into the gel and then free dried at 260C.
This step was repeated 5 times and a multilayer
integrated membrane was obtained into the knitted
structure. These meshes were analyzed by spectroscopic
(FT-IR), mechanical and morphological (water absorption,
permeability and SEM) analyses. In order to evaluate the
collagen matrices the in vitro tests for enzymatic
biodegradability and biocompatibility with endothelial
cells were also performed. Also, after implantation test,
the experimental biomaterials were excised with tissue
for histological and scanning electron microscopy
evaluation.
Results. The polypropylene and polyester-collagen mesh
was very well integrated in the connective tissue, but in
the case of polyester mesh was observed the presence of
inflammatory elements.
Conclusion. The combination of prosthetic biomaterials
with collagen to form various composite meshes will
provide a better biointegration of the mesh in the
surrounding tissue. Scanning electron microscopy appears
as a valuable method in order to establish the
biodegradability degree of the biodegradable structures
used for abdominal mesh.
Keywords. collagen, abdominal tissue, scanning electron
microscopy, biodegradable mesh
(2.P3) A NEW METHOD FOR SELECTIVE OXIDATION OF
HYALURONIC ACID – A VERSATILE, NONTOXIC AND
CROSSLINKABLE MATERIAL FOR TISSUE ENGINEERING
AND REGENERATIVE MEDICINE
Buffa R (1), Hašová M (1), Kettou S (1), Huerta-AG (1),
Dvořáková J (1), Němcová M (1), Velebný V
(1) CPN
Introduction. Hyaluronic acid (HA) is a natural linear
heteropolysaccharide consisting of D-glucuronic acid and
N-acetylglucosamine units, with a molecular weight of 5 13000kDa. High concentrations of hyaluronate can be
found in skin, vitreous humour, cartilage and the
umbilical cord.
Results. The new type of modification of hyaluronic acid
was investigated. The selective oxidation processes
leading to the formation of aldehyde moiety in the
position 6 of N-acetylglucosamine part of hyaluronic acid
dimer were developed and optimised. Two oxidation
agents were successfully tested to perform this
modification. Dess-Martin periodinane (DMP) in DMSO
produced highly substituted derivatives with the degree
of substitution (DS) around 50%. Application of DMP
caused a significant degradation of polysaccharide
resulting the molecular weight around 20kDa (starting
material 1MDa). Second system includes 2,2,6,6Tetramethylpiperidine-1-oxyl (TEMPO)/NaClO in water at
lower temperature. Degree of substitution was circa 15%,
but no significant degradation was observed (1MDa
→
500kDa). The possibilities to form cross-linked materials
were successfully tested using various bis-amino linkers.
The structures of products were elucidated by advanced
NMR
methodologies
and
by
size
exclusion
chromatography SEC-MALLS.
Cell viability was measured using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay to obtain basic information about cell
metabolism. The results had shown that the viability of
cells after HA-Ox (DS 5-50%, Mw 10-500kDa) treatment
was not significantly changed in whole monitored interval
(0-72 hours, Fig. 1).
Conclusion. In this study, the synthesis and the influence
of HA-Ox on 3T3 fibroblast cell line was examined. HA-Ox
did not have any effect on cell viability compared to
untreated control and is safety up to 1mg/ml. Crosslinked
materials prepared from this precursor are biocompatible
and suitable for application in tissue engineering and
regenerative medicine.
Acknowledgement: This research project was conducted
under financial support provided by the Ministry of
Industry and Trade of the Czech Republic.
Keywords. cell viability, oxidation, biomaterial,
polysaccharide
(2.P4) IN VIVO BIOPRINTING FOR COMPUTER- AND
ROBOTIC-ASSISTED
MEDICAL
INTERVENTION:
PRELIMINARY STUDY IN MICE
Keriquel (1), Catros (1), Ziane (1), Bareille (1), Rémy (1),
Rousseau (1), Amédée (1), Chassande (1), Guillemot (1),
Fricain (1)
1. INSERM U1026; University of Bordeaux
Introduction. Bioprinting technologies have emerged
over the last decade to pattern cells and growth factors
within 3D engineered structures in vitro.We recently
demonstrated the feasibility of using bioprinting in situ
and in vivo. This work deals with bioprinting of
mesenchymal
stem
cells
and
hydroxyapatite
nanoparticles into critical size bone calvaria defects of
living mice.
Materials and Methods. Critical size bone calvaria defects
were performed in fifty-six OF-1 male and in Balb/cJ
femelle mice with a 4mm diameter trephine.
Hydroxyapatite nanoparticles (n-HA), prepared by wet
precipitation,
and
luciferase-transducted
mouse
mesenchymal stem cells (D1-Luc cells) were printed
directly into calvaria defects using a workstation
dedicated to high-throughput Laser-Assisted Bioprinting.
Decalcified histology, x-ray microtomography and
bioluminescence (Photon Imager-Biospace) were carried
out to characterize tissue neoformation over 3 months.
Results. Decalcified histology and x-ray microtomography
have shown that in situ bioprinting of nanohydroxyapatite may favor bone healing. Non-invasive
detection, localization and quantification of printed D1Luc cells using bioluminescence have shown cell survival
and proliferation over several weeks.
Conclusion. These preliminary results demonstrate that in
vivo bioprinting is possible and that mesenchymal stem
cells deposited in situ proliferate. Bioprinting may prove
to be helpful in the future for medical robotics and
computer-assisted medical interventions.
Keywords. in vivo, bioprinting, bone tissue engineering
(2.P5) USE OF CEMENT IN ANTIBIOTIC IMPREGNATED IN
SURGERY ARTHROPLASTY INTERACTIVE
Jeice de Souza I (1), Nicodemos da Silva S (1)
1. Department of Materials Engineering, Federal Center of
Technological Education of Minas Gerais (CEFE-MG,
Brazil)
The surgical cement used to secure the prosthesis in the
medullary cavity of the joints is composed of
polymethylmethacrylate (PMMA). This polymer allows
the attachment of the prosthetic device for penetration
into the bone on a metal. The natural wear of the
prosthesis occurs inevitably, leading to its replacement
after about 10 years of use, through a new procedure (or
reintervention) surgery, called interactive arthroplasty.
However, the risk of infection foci of infection is also a
possible cause of exceptional complication, often
requiring a second intervention in order to avoid major
problems, along with use of antibiotics postoperatively.
The infection may appear early after the intervention (10
to 20%). This study was followed by, first, the effect of
adding the antibiotic vancomycin in PMMA
reinterventions made during a public hospital in the
Brazil, noting the short and medium term evidence of
infection. The antimicrobial spectrum of vancomycin is
the treatment of gram positive and anaerobic bacteria.
Through laboratory tests, he noticed the sensitivity of
microbiological patient, realizing that even the additional
protocol of 4 to 6 g of antibiotic in the cement, noted the
emergence of infections, resulting in replacement of the
prosthesis. It was found during reconstruction of PMMA
the temperature reached 100
⁰ C, causing a loss in
biological fixation prosthesis due to cell death in the
cement interface, bone, and the degradation of
vancomycin, reducing its antimicrobial action.
Consequently, using high doses of adjuvant antibiotic for
an extended period of 4 weeks. However, in view of the
high demand of femoral hip implants and the
considerable increase of these failures, we find it
essential to intensify the supervision and standardization
of all procedures involved in order to optimize the
manufacturing process for obtaining a product reliable
health care, particularly in the areas of durability and
biofunctionality.
Keywords. Arthroplasty, impregnation of antibiotic
PMMA
(2.P6) INVESTIGATING THE CONSISTENT SCALED
PROCESSING OF HUMAN EMBRYONIC STEM CELLS
Guijarro-Leach JJ (1), Ratcliffe E (1), Young L (2), Denning
C (3), Williams D (1), Thomas R (1)
1. Centre for Biological Engineering, Wolfson School of
Mechanical
and
Manufacturing
Engineering,
Loughborough University, LE11 3TU, UK; 2. UK Stem Cell
Bank, Blanche Lane, South Mimms, Potters Bar,
Hertfordshire, EN6 3QG, UK; 3. Wolfson Centre for Stem
Cells, Tissue Engineering and Modelling (STEM), The
University of Nottingham, Centre for Biomolecular
Sciences
Human embryonic stem cells (hESCs) are notoriously
difficult to maintain in culture. hESC cultures handled
inappropriately are often phenotypically and genetically
unstable. The analysis of hESC culture quality is
complicated by the variation that exists between
laboratories within the same hESC lines and between
different hESC lines (documented by the ongoing ISCI
project - International Stem Cell Characterisation
Initiative). There is little clarity regarding the extent to
which these differences are intrinsic to the cell lines, the
measurement systems or simply the adaptation to
different culture conditions or platforms.
The culture of hESCs is further complicated by the gold
standard method that produces heterogeneous colonies
in undefined culture media using animal components.
This state of affairs is not tenable as a platform for
regulated therapeutic products where cells of measurable
and reproducible purity and potency from a GMP
compatible production system are regulatory necessities.
If this is ever to be realised, the standardisation of largescale culture systems capable of achieving consistent cell
populations will need to be developed.
This project is driven by the requirements of the project
partner, the UK Stem Cell Bank (UKSCB), to achieve
reproducible and scalable culture methods for the
distribution of stem cells and, builds on the recently
published success from Thomas et al( 2009,
Biotechnology and Bioengineering)demonstrating the
capability of a large scale robotic system (CompacT
Select) at maintaining both pluripotency and a consistent
proliferation rate of hESC lines Hues-7 and Nott-1. The
project aims to further characterize the processing of
hESCs under different culture conditions by systematically
investigating the responses (hESC critical to quality
marker profiles) and interactions between several key
processing parameters, identified through the creation
and analysis of high-detail process maps in an attempt to
determine optimal windows of operation for the
consistent large scale production of high quality hESCs.
Keywords. Human embryonic stem cells, pluripotentcy,
large scale, automation, optimisation, quality, process
control
(2.P7)
POROUS
GELATINE-HYDROXYAPATITE
COMPOSITE SCAFFOLDS VIA GAS-IN-LIQUID FOAM
TEMPLATING
Pecci R (1), Barbetta A (2), Bedini R (1), Dentini M (2)
1. Technology and Health Department, Italian National
Institute of Health, Rome, Italy; 2. Department of
Chemistry, Sapienza University of Rome, Italy
Introduction. Gelatin and hydroxyapatite (HAp) sponges
because of their biocompatibility and biodegradability
have the potential to be used as scaffolds to support
osteoblasts and to promote bone regeneration in
defective areas. In this work gelatine and HAp composites
were fabricated in a foam type via a novel foam
templating technique.
Materials and Methods. A dispersion of nano HAp
particles in a concentrated solution of gelatine and an
appropriate
surfactant
was
foamed
using
hexafluoroethane as the blowing agent. The foam, once
formed, was frozen in liquid nitrogen and then freezedried. Subsequently it was cross-linked with a
carbodiimide derivative to retain its chemical and thermal
integrity. X-ray computed microtomography was used to
nondestructively and quantitatively measure the threedimensional porosity and the morphometric parameters.
The samples were scanned with a Skyscan 1072 µ-CT
imaging system (Belgium) at 7,32 µm resolution and with
following settings: 40 kV and 250 µA. Image
reconstruction and analysis were conducted using the
software package provided by Skyscan.
Results. All the scaffolds synthesised exhibited an
excellent, totally interconnected trabecular morphology.
A content of HAp up to 40 % w/w was achieved. Through
µ-CT it was shown that HAp particles are distributed
homogeneously within the gelatine framework (fig.1). In
order to achieve a higher level of HAp content, similar to
that of natural bone (~ 70% w/w), the composite scaffold
characterised by a HAp content of 40 % w/w was
subjected to four cycles of deposition of HAp on the
scaffold walls. The final content of HAp as determined by
thermogravimetry was very close to 70 % w/w.
Conclusion. The foaming technique described, associated
with the deposition procedure permits the preparation of
scaffold that fulfil both from a morphological and
compositional point of view the main characteristic of
trabecular bone and as a consequence are promising as
constructs for bone tissue engineering.
Keywords. Micro-computed tomography, biomaterials,
bone substitutes, scaffold
(2.P8) LAYER-BY-LAYER BIOFABRICATION USING LASERASSISTED-BIOPRINTING
AND
ELECTROSPINNING
ENHANCES CELL PROLIFERATION IN VITRO AND IN VIVO
Catros S (1), Nandakumar A (2), Ziane S (1), Keriquel V (1),
Moroni L (2), Blitterswijk C (2), Rousseau B (1), Amédée J
(1), Guillemot F (1), Fricain JC (1)
1. Inserm 1026 "Bio Tis", Bordeaux University, France;2.
Department of Tissue Regeneration, University of Twente,
the Netherlands
Introduction. Laser-Assisted-Bioprinting (LAB) is an
effective printing technology for patterning cells,
biomolecules and biomaterials, and electrospinning may
be used to build thin membranes of polymers. The aim of
this work was to associate LAB and electrospinning to
achieve three-dimensional cellularized materials and to
evaluate the influence of layer-by-layer bio-fabrication on
MG63 cell proliferation in vitro and in vivo.
Materials and Methods. The LAB setup comprised an
infra red laser (Nd:YAG 1064 nm, 30 ns) controlled by
scanners, and focused onto glass ribbons coated with a
gold absorbing layer (30 nm). Space between ribbon and
quartz substrate was 400µm. The Polycaprolacton (PCL)
scaffolds (100µm thick) were prepared using a PCL
solution (20% w/v in CHCl3) loaded into a syringe and
electrospun using a pump and a high voltage generator.
MG63 osteoblastic cells transfected with luciferase were
cultured in complete medium (IMDM supplemented with
10% FBS). The concentration of cell bio-ink was 50.106
cells/ml, suspended in 1% alginate solution (w/v) and
culture medium. The building sequence of the test group
comprised three sequential layers of cells and PCL
scaffolds stacked. In the control group, a similar amount
of cells was printed over three PCL membranes stacked.
Then, the materials were cultured in vitro during 3 weeks
or implanted 2 months in bone calvarial defects of 20
NOG mice. Follow-up was done using photon imager
quantification in vitro and in vivo and histological
analyses.
Results. In vitro and in vivo results have shown that layerby-layer bio-fabrication significantly enhanced cell
proliferation. Histological analyses confirmed that the
tissues retrieved after sacrifices were thicker in the layerby-layer group.
Conclusions. We have demonstrated in this model that a
layer-by-layer bio-fabrication using LAB and PCL scaffold
is an efficient combination to improve cell proliferation in
vitro and in vivo.
The authors would like to thank the IFRO, the FRM, the
GIS-AMA and the Aquitaine Region for financial support.
Keywords. Layer-by-Layer; Electrospinning; Laser Assisted
Bioprinting
(2.P9) IN VITRO ENGINEERING OF A TRACHEAL
EPITHELIUM: CO-CULTIVATION OF TRACHEAL EPITHELIAL
CELLS AND FIBROBLASTS ON SMALL INTESTINAL
SUBMUCOSA SEGMENTS
Thome-van de Wal R (1), Haverich A (2), Hilfiker A (1)
1. Leibniz Research Laboratories for Biotechnology and
Artificial Organs (LEBAO), Hannover, Germany; 2.
Department of Cardiac, Thoracic, Transplantation and
Vascular Surgery, Hannover Medical School, Hannover,
Germany
Introduction. Surgical correction of large tracheal defects
remains to be a tackling problem. Lesions that cannot be
treated by an end-to-end anastomosis and need an
interponate which often fail to regenerate a functional
tracheal epithelium. Here we investigated whether
suturable decellularized small intestine submucosa (SIS)
may serve as matrix for the in vitro generation of tracheal
epithelium.
Materials and Methods. Primary tracheal epithelial cells
and fibroblast were harvested from porcine trachea by
Protease XIV and Collagenase A digestion, respectively,
and cultured in their appropriate culture media. For
seeding purposes decellularized SIS, generated from
porcine small intestine by decellularization, was clamped
in stainless steel frames. Primary isolates of epithelial
cells were seeded onto the sub-mucosa side of the SIS
after reaching 80% of confluency in culture flasks.
Stimulatory effects of tracheal fibroblasts were tested by
seeding cells onto the sub-serosa side of the SIS
(constructs without fibroblast served as controls). SEM
and Histology analysis of constructs were conducted after
five days of culture of which three were spent as air liquid
interface culture.
Results. SEM examination and Phalloidin stains show a
completely covered SIS with orientated respiratory
epithelium. Immunohistochemistry against Cytokeratin
14 (basal cell marker), Mucin 5AC (goblet cell marker) and
β-Tubulin IV (ciliate cell marker) demonstrated a
pseudostratified-like epithelium. The production of
glycosaminoglycan and Mucin 5AC was more pronounced
after fibroblast co-culture.
Conclusion. Decellularized SIS is suited for culturing
tracheal epithelium and may serve as useful matrix for
tracheal tissue engineering purposes for the generation of
surgical implants.
Keywords. tracheal epithelium, tissue engineering, SIS, air
liquid interface culture
(2.P10) BIOFABRICATION OF TISSUE ENGINEERED
VASCULAR GRAFTS
Elsayed Y (1), Bi Z (1), Lekakou C (1), Tomlins P (2)
1. University of Surrey; 2. National Physics Laboratory
Cardiovascular disease is the largest contributor to
mortality in the world claiming nearly 30 percent of all
deaths. Tissue engineered scaffolds are essential for small
diameter vascular grafts to avoid the fatal risk of
thrombosis of the synthetic vascular grafts. In this work,
biomimetic gelatine/elastin fibrous scaffolds are
proposed, fabricated by electrospinning as tubular
constructs. Tissue engineering then takes place in vitro in
a bioreactor, in which the tubular scaffold is rotated in a
bioreactor surrounded by a smooth muscle cell (SMC)culture medium suspension, while a suspension of
endothelial cells (ECs) flows axially inside the tubular
scaffold in a recirculating flow. A novel fluorescence
quenching type of sensor has been developed to be
embedded at different positions in the scaffold for
continuously monitoring the oxygen concentration in the
growing tissue. Adherence, growth and proliferation of
both types of cells is examined for different scaffold
structures and different processing conditions, such as
cell concentration, flow rate of the cell-culture medium
suspension and rotation speed of the scaffold. The fibrous
scaffolds have been crosslinked using glutaraldehyde as a
crosslinking agent. Cytotoxicity studies are also carried
out to investigate the effect of glutaraldehyde on the cell
growth and proliferation.
Keywords. Scaffold, Fluorescent quenching, bioreactor,
vascular graft
(2.P11) FABRICATION OF THREE-DIMENSIONAL CELLLADEN HYDROGEL FOR SOFT TISSUE ENGINEERING
Park SA (1), Lee SH (1), Lee JH (1), Kim WD (1)
1. KIMM, Republic of Korea
Three dimensional (3D) scaffolds should be porous to
transfer oxygen and nutrient for cell proliferation and
differentiation in tissue engineering. Scaffolds have been
fabricated using various conventional techniques of salt
leaching, freeze drying, fiber bonding, phase separation,
and gas expansion. However, they have a limitation of
homogeneous cell distribution on the scaffold. Scaffold
fabrication techniques need to control 3D pores inside
scaffold. In these methods, solid freeform fabrication
(SFF) of rapid prototyping (RP) technology has been
adopted to 3D scaffold design with controllable and
reproducible porosity and well-defined 3D structures for
tissue engineering. Especially, soft tissue has a very high
content of water, so scaffolds need a hydrogel material.
Hydrogel biomaterials can provide the micro environment
to build up by living cells and the extracelluar matrix
(ECM) due to their structural similarities to the body
tissues, biocompatibility and low toxicity. In this study, we
manufactured 3D scaffold plotting system (SPS) to design
interconnected scaffold and fabricated interconnected
hydrogel scaffold with cells through plotting process and
developed the software using the geometrical data
obtained from stereolithography (STL) file format for SPS
operation. Also, we fabricated cell-laden hydrogel
scaffold including gelatin to help cell growth. 2% alginate
with cells was plotted under various pressure conditions
of SPS system. Cell-laden alginate hydrogel had a regular
cell distribution and a good cell viability in vitro test. We
confirmed the potential of the 3D hydrogel scaffold for
soft tissue engineering application.
Keywords. scaffold, fabrication, hydrogel, soft tissue
engineering
(2.P13) DEVELOPMENT OF A NEW TECHNIQUE FOR
MUSCLE TISSUE-STEM CELLS CO-CULTURE
Erratico S (1), Belicchi M (2), Razini P (2), Farini A (2),
Meregalli M (2), Villa C (3), Torrente Y (2)
1. Stem Cell Laboratory, Fond. IRCCS Ca’ Granda Ospedale
Maggiore Policlinico, Università di Milano, Fond. Filarete,
PhD School Molecular Medicine; 2. Stem Cell Laboratory,
Fond. IRCCS Ca’ Granda Ospedale Maggiore Policlinico,
Centro Dino Ferrari, Università di Milano; 3.Stem Cell
Laboratory, Fond. IRCCS Ca’ Granda Ospedale Maggiore
Policlinico, Centro Dino Ferrari, Università di Milano, Fond.
Filarete, SEMM
Introduction. Adult stem cells reside in all tissues, where
they maintain homeostatic conditions and respond to
injuries. These cells are regulated and supported by the
surrounding microenvironment, called stem cell “niche”,
composed by cellular and molecular factors, that interact
with and regulate stem cell fate. Key niche components
are represented by growth factors, cell-cell interactions
and cell-matrix adhesion. Also in muscle tissue niches are
present, especially in myofiber basal lamina, where a
network of extracellular matrix components and secreted
growth factors stimulate muscle stem cell survival,
activation and/or proliferation. In order to better
understand the influence of muscle tissue secreted
factors on stem cells, we developed a new technical
approach to perform a muscle tissue-stem cells coculture; we focused on peripheral blood derived CD133
cells, a population known to possess a myogenic
potential.
Materilas and Methods. Muscle tissue sections were cut
with a tissue chopper from fresh quadriceps of C57BL
mice and inserted in a culture well upon a porous
membrane, above cells suspension; cells were isolated
through
immunomagnetic
separation
column;
immunophenotypic characterization was performed with
Cytomics FC500.
Results. The culture system developed creates a physical
separation between tissue section and stem cells,
allowing soluble factors exchange and preventing tissuecell contamination; the absence of cell mix was evaluated
through cytogenetic analysis of cell karyotype; the
reproducibility of the technique has been demonstrated
through muscle slice weight monitoring. Preliminary
proliferation experiments show an increase in CD133+
stem cells rate of proliferation in presence of muscle
tissue; cell immunophenotype monitoring confirmed
stemness maintenance.
Conlcusiones. We have developed a reproducible and
standardized technique and designed a culture system
that guarantees, spatial division of culture environment,
no
cellular
contaminations
between
culture
compartments, in vitro tissue survival, stem cell viability
and proliferation, communication between tissue and
cells through chemical signals (factors release).
Keywords. stem cells; co-culture; muscle tissue
3. BIOFUNCTIONAL MATERIALS AS
EXTRACELLULAR SIGNALS TO
PROMOTE TISSUE MORPHOGENESIS
Chair: Elisabeth Engel
Co-chair: Josep A. Planell
Keynote speaker: Abhay Pandit
Organizers: Josep A. Planell, Elisabeth Engel
Synopsis: Regenerative medicine based on tissue
engineering needs a step forward in biomaterials design
coupled with a search for novel activities and evaluation
of their behaviour in biological systems. The body’s
capacity to regenerate it is not well elucidated but several
signals implicated in regeneration have been revealed
already. Among all of them, signals connected to adult
stem cells mobilization to the injury site and activation of
the repair scheme as well as new tissue formation are the
most relevant. The ability to direct stem or progenitor cell
differentiation via a chemically/naturally synthesized
biomaterial, without the need to incorporate growth
factors or other molecules that might induce undesirable
effects, offers many potential advantages in regenerative
medicine. The properties of the own materials are the
ones that stimulate cells to produce the appropriate
chemokines and growth factors to promote cell
activation. This activation can be the mobilization of stem
cells out of its niche to go to the injured tissue. At the
injured site, cells will produce the molecules to induce
tissue repair. For example, ion release can induce this
mobilization and call the progenitors to the implant site.
Besides chemical signalling we have to take into account
the physical signalling to induce the most appropriate
response of the surrounding cells. Surface topography has
been demonstrated to have an effect in several biological
activities, as cell adhesion, migration, proliferation and
differentiation. But we cannot forget the mechanical
properties of the biomaterials, as it has been already
demonstrated. The surface stiffness plays a definite role
in stem cell differentiation, when mimicking the tissue
stiffness. Thus, inflammation is also a mechanism in tissue
repair. The use of biomaterials that could modulate
inflammatory responses to avoid chronic inflammatory
responses (characterized by leukocyte adhesion and
fibrous encapsulation) but promoting a signalling cascade
that will induce tissue formation is also a major issue in
tissue engineering. This holistic view will be the next
generation of biomaterials to be applied in advanced
therapies to treat diseases related to tissue degeneration.
The properties of the biomaterials will conduct the own
body repair.
(3.KP) A FUNCTIONALISED SCAFFOLD FOR MODULATION
OF INFLAMMATION TO PERMIT STEM CELL SURVIVAL IN
MYOCARDIAL INFARCTION
Pandit A (1)
1. Network of Excellence for Functional Biomaterials,
National University of Ireland, Galway
Cardiovascular disease is the leading cause of death in the
developed world and is responsible for approximately
36% of Irish mortality. Myocardial infarction (MI), which is
literally the death of cardiac tissue due to lack of
oxygenation, accounts for the majority of deaths
associated with cardiovascular disease. This death of
cardiac tissue leads to a loss of cardiac function as the
damaged area becomes a non-contractile scar. Reversal
of this process is the main aim of regenerative cardiac
strategies such as stem cell transplantation. While initial
studies were promising, subsequent clinical trials yielded
disappointing results. Stem cell therapy may be limited by
the poor survival rate of the cells after implantation into
the infarcted heart, which is likely due to the
inflammatory response. Thus, anti-inflammatory gene
therapy with interleukin-10 (IL-10) was proposed as a
method to modulate the inflammatory response after
implantation of a collagen scaffold seeded with rat
mesenchymal stem cells (rMSCs). IL-10 is considered the
most potent anti-inflammatory cytokine produced
naturally and has been used in a number of studies to
decrease or control inflammation. It was hypothesized
that IL-10 gene therapy could be used to increase the
retention rate of stem cells in a collagen scaffold when
delivered to the ischemic myocardium. The primary
objectives were to develop a controlled release scaffoldbased gene therapy system suitable for stem cell delivery
to the infarcted myocardium. The efficacy of this system
was evaluated by assessing stem cell retention, overall
cardiac function and the inflammatory response. A
crosslinked collagen scaffold was developed and
optimised for rMSC culture in vitro. Non-viral plasmiddendrimer polyplexes were optimized for transfection in
both two and three-dimensional culture. When cells were
seeded into polyplex loaded scaffolds, relatively high
levels of transgene expression were observed for up to
three weeks of culture. When the polyplex-loaded
scaffolds were implanted in rat skeletal muscle, increased
retention of rMSCs was observed. This was associated
with decreased inflammation and a change in
macrophage phenotype from cytotoxic to regulatory.
Similarly, when the polyplex-loaded scaffolds were
implanted over the surface of infarcted rat hearts, rMSC
retention was increased, the inflammatory and
remodelling responses were modulated and, most
importantly left ventricular ejection fraction – a measure
of cardiac function – was significantly improved. Thus,
combining biomaterial, gene and cell therapy improved
functional outcomes after rMSC transplantation following
MI. This combinatorial strategy can be utilised to provide
functional efficacy in disease targets.
(3.O1) EVALUATION AND PREDICTION OF ACUTE
INFLAMMATORY CHARACTERISTICS OF IMPLANTABLE
SYNTHETIC AND TISSUE-BASED BIOLOGIC MESHES
USING A SENSITIVE QUANTITATIVE IN VITRO
CHEMILUMINESCENT ASSAY
Bryan N (1), Bayon Y (2), Scarborough N (3), Hunt J (4)
1. University of Liverpool; 2. Covidien - Sofradim
Productions; 3. Covidien; 4. University of Liverpool
Clinical performance and therapeutic outcome of mesh
assisted soft tissue augmentation is decided early after
implantation as leukocytes interrogate the graft in the
first day postoperatively. High degrees of leukocyte
activation lead to chronic pain. Reactive oxygen species
(ROS) are released by leukocytes when activated. This
response can be used as a sensitive measurement of
leukocyte activation. The aim of this study was to
compare the degree of leukocyte activation of
commercially available synthetic and biological meshes.
Materials and Methods. Chemiluminescence assay was
performed using modifications to a commercially
available kit (Knight Scientific, UK). Whole blood was
obtained from 5 different healthy human adults,
combined with Adjuvant K, Pholasin and graft, and
incubated
for
30
minutes
with
continuous
chemiluminescent measurements. Leukocyte stimulants
fMLP and PMA were added as controls. Synthetic meshes
of varying chemistry (PP, PET, PGA) and knitting patterns
and xeno- and allogeneic dermis and small intestinal
submucosa (SIS) biological meshes prepared with varying
decellualrisation techniques. Statistics were performed
using Waller-Duncan post hoc ranking into statistically
homogenous subsets (Fig.1).
Results. Chemiluminescence measurements of ROS
demonstrated material specific differences in leukocyte
activation. Among synthetic meshes, multifilament PGA
mesh had significantly higher responses compared to PP
and PET meshes (p<0.05). Yarn conformation (ie. monovs multi-filament) made a greater difference to the
leukocyte response than polymer composition. WallerDuncan post hoc ranking allowed grouping of the
materials into statistically similarly ROS stimulating
groups.
The biological meshes demonstrated significant
differences in leukocyte activation as a function of
decellularisation reagent and related tissue origin, the SIS
mesh and SDS decellularisation strategies eliciting the
greatest stimulation.
Conclusion. The most leukocyte activating synthetic and
biological meshes were the Multifilament PGA mesh and
the SIS mesh, respectively. In the case of synthetic
meshes it was concluded that weave is a greater
influence on leukocyte response than polymer chemistry.
Keywords. In Vitro, Leukocyte, Inflammatory Response,
Hernia
(3.O2) IMPROVEMENT OF BIOLOGICAL PROPERTIES OF
POLYMERIC
MATERIALS
THROUGH
THE
BIOFUNCTIONALIZATION WITH ELASTIN-LIKE POLYMERS
Punet X (1), Mauchauffé R (1), Engel E (1), RodríguezCabello JC (2), Mateos-Timoneda MA (1), Planell JA (1)
1. IBEC; 2. Bioforge UVA
Introduction. In tissue engineering, scaffolds made up of
synthetic polymers are usually selected because they fit in
many aspects the requirements of biomedical materials,
such as biocompatibility, biodegradability, malleability.
However, these synthetic polymers lack bioactivity, i.e.
they do not present groups or moieties that guide the
interactions between materials and cells, which difficult
the implementation of such devices in the biomedical
field. In order to improve that aspect, approaches based
on the incorporation of active biomolecules on the
material surface have been widely investigated. One of
the most known and used biomolecule is the RGD peptide
sequence, which has been implemented on surfaces in
the form of short peptide. This report proposes the
incorporation of the RGD sequence through the
functionalization of the biodegradable polymeric surface
with an elastin-like polymer (ELP) that includes the RGD
inside the amino acidic chain. The ELPs are a genetically
modified version of the natural elastin. The natural origin
gives to the ELP constructs mechanical properties that are
not found on short peptides and a more natural cell
environment. The report compares the cell response
against surfaces of poly(lactic acid) functionalized with
ELPs and short peptides. Special emphasis has been put in
the comparison of covalent functionalization against the
physisorption. Also an ELISA-based assay is proposed for
the quantification of peptides on surface.
Materials and Methods. Functionalization of surfaces is
obtained through the creation of amide bonds with the
EDC/NHS chemistry. Cell response against the different
treated surfaces is studied through the quantitatively
analysis of the cell adhesion capacities and cell
proliferation. For the quantification of grafted molecules,
a PEG-biotin molecule is used as an analogue of a peptide
molecule in order to draft the design of the assay.
Results and Conclusion. The enhancement of the
material biological properties through the ELP
functionalization has been proved to be higher than the
enhancement obtained with the short peptides. The
ELISA-based assay has proved to be able to quantify the
amount of biomolecules on surface.
Keywords. biofunctionalization, cell-material interaction,
tissue engineering
(3.O3) MOLECULAR MECHANISM INVOLVED IN THE
WOUND HEALING EFFECT OF SILK PROTEINS FIBROIN
AND SERICIN
Martínez-Mora, C (1), Mrowiec, A (2), Alcaraz, A (2),
López-Martínez, C (2), Aznar-Cervantes (1), GarcíaVizcaíno, E (3), Cenis, JL (4) Nicolás, FJ (4)
1. IMIDA, Murcia, Spain; 2. Oncología Molecular y
TGFbeta, Hospital Universitario Virgen de la Arrixaca,
Murcia, Spain; 3. Oncología Molecular; 4. IMIDA; Murcia,
Spain; Oncología Molecular y TGFß, HUVA, Murcia, Spain
Introduction. Wound healing is a biological process
directed to the restoration of tissue that has suffered an
injury. An important phase of wound healing is the
generation of a basal epithelium able to wholly replace
the epidermis of the wound. A broad range of products
derived from Fibroin and Sericin is used to stimulate
wound healing. However, so far the molecular
mechanism of this phenomenon has not been
determined. Fibroin is a protein secreted by the silkworm
Bombyx mori and has unique properties such as good
biocompatibility, lack of immune response and
biodegradability. Sericin is the second main silk protein,
being the sticky material surrounding Fibroin fibers. The
aim of this work is to determine the molecular basis
behind wound healing properties of silk using a cell
culture model.
Materials and Methods. For this purpose, we assay
Fibroin and Sericin in a wound healing scratch assay using
Mv1Lu and MDA-MB231 cells. Both proteins stimulate
cell migration. Furthermore, treatment with Sericin and
Fibroin regulates key factors of the wound healing
process upregulating c-jun gene expression and c-Jun
protein phosphorylation. Moreover, Fibroin and Sericin
stimulates the phosphorylation of SAP/JNK kinase and
phosphorylation of ERK 1 and 2. All these experiments
were done in the presence of specific inhibitors for some
of the cell signalling pathways referred above.
Results and Conclusion. The obtained results revealed
that only the inhibitors of SAP/JNK kinase, but not p38,
PI3K or ROCK inhibitors prevent cell migration stimulated
by Fibroin or Sericin.
Keywords. Fibroin, Sericin, Wound Healing, Silk
(3.O4) DYNAMIC SURFACES TO INFLUENCE STEM CELL
DIFFERENTIATION
Roberts, JN (1), Burchmore, RJ (1), Ulijn, RV (2), Dalby, MJ
(1)
1. University of Glasgow; 2. University of Strathclyde
Introduction. Stem cell differentiation is governed by a
series of complex intracellular signalling pathways. One of
the main pathways, the ERK1/2 mitogen activated protein
kinase pathway relies on integrin dependent cell-surface
interactions to trigger a cascade resulting in changes to
gene transcription and expression. The intention of this
work is to synthesise a functionalised surface capable of
switching from a state that does not actively promote cell
adhesion “off” to a state that readily promotes cell
adhesion “on” and hence dynamically influence stem cell
differentiation. This is achieved using Fmoc (9Fluorenylmethoxycarbonyl) protected amino acids to
build up the desired peptide chain on the substrate
(Figure 1). Here the peptide sequence is RGD, a known
integrin binding peptide sequence. On demand switching
is achievable by enzymatically removing the terminating
Fmoc group of a short cleavable peptide which, when
attached, acts to prevent cells from interacting with the
underlying integrin recognition motif. The long-term aim
of this project is to synthesise a surface that can be
enzymatically switched by the cells as they run out of
room to proliferate and begin to differentiate thus
allowing synchronised progression directed by the
dynamic material surface.
Method and Materials. Surfaces were synthesised using a
method described by Todd et al 2009 [1]. After surface
modification, Human mesenchymal stem cells (hMSCs)
were cultured directly onto surfaces containing both the
“bio-active” RGD and non “bio-active” RGE forms in both
the cleaved (“on” state) and non-cleaved (“off”) states
(Figure 1). After a period of 7 days cell spreading and cell
morphology were quantitatively investigated using
fluorescence microscopy by staining for actin and vinculin
alongside nuclear staining. Focal adhesions were analysed
by tracing adhesion outlines and using Image J software
to determine numbers and lengths.
Results and Conclusion. For the cells to respond to the
“switching on” of RGD groups through enzymatic
cleavage of Fmoc, we have derived optimal media
conditions. In these conditions, as Fmoc is cleaved, the
MSCs rearrange their adhesions increasing numbers of
larger adhesions. The formation of large adhesions is
important for osteogenesis through support of increased
intracellular tension. Future work will focus on balancing
MSC proliferation and differentiation through dynamic
surface properties [2-3].
With thanks to the EPSRC
References:
[1] Todd SJ, Scurr DJ, Gough JE, Alexander MR, Ulijn RV.
Enzyme-Activated RGD Ligands on Functionalized
Poly(ethylene glycol) Monolayers: Surface Analysis and
Cellular Response. Langmuir 2009;25(13):7533-7539.
[2] Kilian KA, Bugarija B, Lahn BT and Mrksich M.
Geometric cues for directing the differentiation of
mesenchymal stem cells. PNAS 2010;107(11):4872-4877
[3] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix
Elasticity Directs Stem Cell Lineage Specification. Cell
2006;126(4):677-689.
Keywords. Stem Cells, Bioactive Surfaces, RGD, Integrins
Figure 1: (A) Representation of proposed surface
chemistry. When the terminating Fmoc group is in place
the RGD sequence is hidden (L). Enzymatic digestion of
the cleavable linker with elastase exposes the underlying
sequence to the cells (R). R groups are Aspartic acid:
CH2COOH, Glutamic acid: CH2CH2COOH and Arginine:
(CH2)2C(NH)2NH2. (B) Human Mesenchymal stem cell
cultured on cleaved surfaces with the “bio-active”
sequence RGD and (C) Human Mesenchymal stem cell
cultured on cleaved surface containing the non “bioactive” sequence RGE. Focal adhesions for both cells are
highlighted in pink.
(3.O5) MUSCLE GRAFT OPTIMISATION PRIOR TO
IMPLANTATION: SCAFFOLD ARCHITECTURE AND
FUNCTIONALISATION INFLUENCE CELL DIFFERENTIATION
Guex G (1), Fortunato G (1), Körner E (1), Carrel TP (2),
Tevaearai HT (2), Giraud MN (2)
1. Inselspital; Empa; 2.Inselpital
Introduction. Cell therapies and associated paracrine
effects for heart regeneration have gained increasing
interest. Epicardial implantation of engineered musclegrafts has been associated with prolonged functional
recovery of the ischemic hearts. These observed effects
are expected to originate from the cell secretion of
cardioprotective, angiogenic or stem cell recruiting
factors or by local delivery of these factors via
functionalisation of the implanted scaffold. In the present
study we performed in vitro studies to investigate the
effects of scaffold architecture and surface
functionalisation on muscle graft development and
related cytokine secretion.
Materials and Methods. Aligned and randomly oriented
micron- (3.2±0.8um) or nano- (308±178nm) scaled fibrous
polycaprolactone non-wovens were processed by
electrospinning. A 15 nm thick oxygen functional
hydrocarbon coating was deposited at the surface by an
RF plasma process (gas mixture: CO_2:C_2H_4 ratio 6:1;
power input: 50 W; process duration: 20 minutes) and
characterised by XPS. C2C12 muscle cells were grown on
the matrices and analysed for viability, proliferation,
orientation and myotube formation. Cell orientation was
characterised by a cosine function, where S=1 for aligned
and S=0 for randomly oriented cells. Cytokine secretion
was assessed using antibody arrays.
Results. The formation of a stable plasma polymer
coating resulted in an 8-14% increased oxygen content on
the matrix. On all scaffolds, cell viability varied from 40 to
60% relative to TCPS. Architectural cues highly influenced
cell orientation. On aligned fibres, cells were highly
oriented (S=0.88±0.02) as compared to randomly
oriented fibres (S=0.33±0.2). Increased myotube
formation was found on CO_2/C_2H_4 coated scaffolds.
Graft contractility and cytokine secretion are under
evaluation.
Conclusion. We provide evidence that the combined
application of architectural and chemical cues is most
favourable for advanced muscle development. Fibre
alignment and plasma coating induced most pronounced
cell
differentiation.
Ongoing
cytokine
release
identification will further characterise this biograft and its
possible promise for cardiac regeneration.
Keywords. electrospinning, plasma coating, muscle
differentiation
(3.O6) EFFECT OF LINE PATTERNED CHITOSAN ON
CORTICAL NEURAL CELLS
Mattotti M (1), Delgado L (2), Planell JA(1), Conrado A (2),
Alcántara S (3), Engel E (1)
1. Institute for Bioengineering of Catalonia-IBEC,
Barcelona, Spain; 2. Dpt. Material Science and
Metallurgical Engineering, Thechnical University of
Catalonia-UPC, Barcelona, Spain; 3. Dpt. of Pathology and
Experimental Therapeutics, Medical School (Bellvitge
Campus), University of Barcelona-UB, Barcelona, Spain
Introduction. Topographical cues have a direct effect on
cell guidance and differentiation. After a lesion in the
central nervous system is necessary to promote a
regenerative permissive environment that allows
replenishment of lost neurons and that guide
regenerating axons to their appropriate targets. In this
study we considered Chitosan (Ch), a biodegradable
biocompatible material carrying good neuronal adhesive
properties. We cultured neurons and glial cells on
uncoated Ch films and we assessed the effect of line
patterns in terms of cell differentiation and orientation by
western blot and immunocytochemistry.
Materials and Methods. Patterned Ch films were
obtained casting a 2% Ch solution on micro-grooved
moulds (2 and 10 µm wide,1 µm deep). 2% Ch films were
highly hydrophilic (Contact Angle 34º±3), positively
charged (Z potential 15±3mV) and had high water
absorption (128±10%.). From the mechanical point of
view, Ch films were quite soft and elastic, having the
following values: Young Modulus 5.7±1.4 MPa, Elongation
at break 56±14% and Tensile Strenght 4.31±0.8 MPa.
Results. Ch films supported good neuronal and glial
growth and the presence of micropatterns induced
alignment. In the case of neurons, alignment was
selective for axons but not for dendrites. Axons on 2µm
lines followed single channels, while on 10µm lines, axons
form bundles, mimicking their physiological 3D structure.
In the case of glial cells, alignment involved the
cytoskeleton and not the whole cell shape. Biochemically,
flat and micropatterned Ch films promoted a general
maturation of glial cells, resulting in an increase of the
mature astrocyte marker GFAP and a decrease of the
immature astrocyte markers BLBP and Nestin, while they
didn’t alter the protein expression of neurons.
Conclusion. Uncoated Ch films promoted neurons and
glial cells attachment and maturation. The in vivo
regenerative ability of Ch scaffolds will be assessed
implanting them into the brain of neonatal mice.
Keywords. chitosan, micropattern, nerve regeneration
(3.O7) RESPONSE OF NEURAL CELLS TO DIFFERENT TYPES
OF POLYLACTIC ACID
Álvarez Z (1), Castaño O (1), Planell JÁ (1), Alcántara S (2),
Engel E (1)
1. Institute for bioengineering of Catalonia; 2. University
of Barcelona
Introduction. The tissue engineering approach to improve
nerve regeneration after an injury is to implant materials
that trigger a regenerative response in situ, promoting a
favorable environment. This project explores the
potential of using Poly L/DL Lactic Acid (PLDLA), an FDA
approved biocompatible and biodegradable polymer, as
scaffold for nerve tissue engineering. PLDLA 95/5 and
70/30 were used in this study, which contain different
proportions of the isomers D and L, have different
cristallinity, degradation rate and surface roughness.
Materials and Methods. PLDLA 95/5 and 70/30 films
were obtained by solvent casting. Embryonic day 16
neurons (E16) and post natal day 0 (P0) glial cells from
mice cerebral cortex were seeded on uncoated PLDLA
films,
cultured
for
5div
and
analyzed
by
imunocytochemistry and western blot.
Results. Both neurons and glial cells attached on PLDLA
70/30 and on 95/5, but neurons attached with more
affinity on 70/30. In addition, PLDLA70/30 induced a
more undifferentiated phenotype of both type of cells.
Glial cultures on PLDLA 70/30 expressed high levels of
Nestin, BLBP and PH3, markers of proliferating radial glia
progenitor cells, while in neuronal cultures increased
Pax6 and Tbr2 markers, characteristic of radial glia
progenitor cells and neuron restricted progenitors.
Conclusion. This study showed that neurons and glial cells
grow on uncoated PLDLA films. PLDLA70/30 was a better
substrate than PLDLA95/5 for neural cells growth and
promoted an environment rich in progenitor cells. Those
results suggest that differences in the proportion of the
isomers D and L in the same polymer can induce different
responses and that PLDLA70/30 could be a good material
for implantation, since it could trigger an in situ
regenerative response.
Keywords. brain, scaffold, tissue engineering
(3.O8) USE OF ACELLULAR WHOLE PIG LUNG AS A
SCAFFOLD FOR STEM CELL BASED PRODUCTION OF
ENGINEERED LUNG TISSUE
Cortiella J (1), Melo E (2), Niles J (1), Nichols JE (1)
1. University of Texas Medical Branch; 2. Unitat de
Biofísica i Bioenginyeria
We report here the first attempt to produce and use
whole acellular pig lung as a matrix to support
development of engineered lung tissue from murine
embryonic stem cells (mESC), pig mesenchymal stem cells
or human amniotic fluid mesenchymal stem cells. Using a
combination of freezing, use of deinonized water and 1%
SDS washes administered through both the trachea and
the pulmonary artery twice daily for two weeks, four
intact pig trachea-lungs were decellularized. Once
decellularization was complete we evaluated the effects
of our decellularization process on the structural integrity
of the lung using two photon microscopy, biochemical
assessment of the extracellular matrix and pulmonary
function tests (PFTs). Two photon microscopic
examinations of trachea and lung tissues showed no cell
remnants but some changes in collagen and elastin
content as decellularization progressed. Biochemical
evaluation of the pig trachea-lung indicated some loss of
type IV collagen but retention of elastin and collagen I.
PFT measurements of the trachea-lungs showed normal
work of breathing, and a non restricted flow pattern.
Analysis of the decellularized tissues did not indicate that
significant levels of unfragmented DNA remained in the
acellular pig trachea-lungs. Although there were some
changes in extracellular matrix they were not significant
as evidenced by low normal PFTvalues. When
repopulated with bone marrow derived mesenchymal
stem cells (MSC) or murine embryonic stem cells (mESC)
the scaffold supported cell attachment and site specific
differentiation. Repopulation of this matrix was similar to
what we have the previously described (1) using rat
trachea-lung as scaffold. Bone marrow derived pig MSC or
mESC cells cultured for 21 days expressed lung cell
specific phenotypes such as surfactant protein C, Clara
Cell protein 10 and thyroid transcription factor -1.
Keywords. engineered lung, acellular scaffold
(3.O9) SURFACE PATTERNING IN STEM CELL
DIFFERENTIATION
Tan Lay Poh (1), Tay Chor Yong (1), Yu Haiyang (1)
1. Nanyang Technological University, SIngapore
While soluble factors has been the classical method to
direct stem cell differentiation, there are growing
evidences illustrating the potential of physical cues such
as surface properties and matrix stiffness in doing the
same. In our work, micro-patterns as big as 20μm lanes to
as small as 3μm squares were created on polymer films
were created on the surface of polymers to trigger
specific human mesenchymal stem cells (hMSCs)
differentiation.
Stem
cells
differentiation
was
characterized by qPCR and immunostaining.Cells cultured
on the lane patterns assume highly elongated and spindle
shape. Gene expression analysis revealed up-regulation of
markers associated to neurognesis and myogenesis while
osteogenic markers were specifically down-regulated.
However at the functionally relevant level of protein
expression, the myogenic lineage is dominant within the
time scale studied as determined by the exclusive
immuno-detection of cardiac myosin heavy chain for the
micropatterned cells. On smaller patterns, the cellular
shape change was less defined but the focal adhesions
(FAs) showed strong correlation to the patterns. The FAs
were regulated into dense and elongated patterns when
the micro-patterns were of small square (3.6X3.6 μm) and
rectangular (2.5X20 μm) shapes respectively. The
synergistic effect of FAs regulation and matrix stiffness
was also explored. The results indicated that dense FAs
would not induce myogenesis while elongated FAs could
promote cytoskeleton alignment and further myogenesis
on PDMS substrate with intermediate stiffness of 12.6
kPa at both mRNA and protein level. But on stiff substrate
(308 kPa) with or without patterns, the cytoskeleton
alignment and myogenesis was not obvious. This work
demonstrates for the first time that it is possible to
induce hMSCs differentiation by regulating the FAs
without any biochemic
We would like to acknowledge Singapore Stem Cell
Consortium (SSCC) (Grant no: SSCC/09/017 for financial
support.
Keywords. micro-patterning, focal adhesion, cell shape,
stem cell differentiation
Immunostaining of hMSCs on PDMS at 7th day of culture.
(A) and (B) were S3.6 patterned substrates. (C) and (D)
were L20 patterned substrates. (E) and (F) were nonpatterned substrates. F-actin (), vinculin (), DAPI labeled
nuclear () were overlaid. For the patterned groups (A-D),
COLI patterns were labeled with Cy3 (). Scale bars showed
50 μm.
(3.O10) NOVEL PEPTIDE-BASED SCAFFOLDS CARRYING
HEPARIN-DERIVED SIGNALS FOR TISSUE REGENERATION
Mammadov R (1), Mammadov B (1), Toksoz S (1), Aydin B
(2), Tekinay AB (1), Guler MO (1), Yagci R (3)
1.
UNAM-Institute
of
Material
Science
and
Nanotechnology, Bilkent University; 2. Medical School,
Mersin University; 3. Medical School, Fatih University
Extracellular matrix (ECM) is a reservoir of signals for
tissue regeneration and repair. These signals can be in
different forms like growth factors, glycosaminoglycans
(GAGs) or bioactive peptide motifs from structural
proteins such as fibronectin or laminin. Although
discovery of GAGs (e.g. heparin) goes to century ago,
their critical role in regulation of stability and
functionality of many growth factors in ECM has been
identified in the last two decades. In this manner,
designing GAG-mimetic scaffolds for tissue regeneration
studies might improve therapeutic efficiency of
biomimetic scaffolds, while allowing to get similar
physiologial output with lower doses of exogenous
growth factors.Taking these into consideration, we
designed Heparin-mimetic peptide amphiphile (PA)
molecule which can be tuned to form ECM-like gel.
Physical characterizations of novel PA scaffolds were
performed by using SEM, AFM and rheology, which shows
similarity to previously designed PA gels. We identified
that novel PA molecule is highly affine to VEGF, which is
heparin-binding growth factor and takes critical role in
angiogenesis. In vitro angiogenesis data shows that
Heparin-mimetic PA matrices induce endothelial cells to
form tube-like structures, similar to Matrigel (basement
membrane gel). Tube formation is accompanied with
increase in expression of angiogenic genes. In vivo studies
further strengthened bioactivity of novel PA scaffolds in
terms of angiogenesis. PA scaffolds with heparin-mimetic
functionalities shown here are promising candidates for
improved regenerative therapies.
Keywords. Angiogenesis, heparan sulphates, peptide
amphiphile scaffolds, peptide gel, biomimetic materials
Acknowledgment. We thank Swedish Research Council
(VR) for financial support.
1. Thorres L. et al., Biomaterials. 29:75-85, 2008
2. Lv S. et al., Nature. 465:69-73, 2010
Key words: autologous scaffolds, macroporosity, skeletal
muscle tissue, myoblasts
Figure 1. Heparin-mimetic PA scaffold induced human
umbilical vein endothelial cells (HUVEC) to form vessellike structures, similar to Matrigel, while non-bioactive PA
scaffold didn’t show any bioactivity different than tissue
culture plate.
(3.O11) AUTOLOGOUS SCAFFOLDS FOR SKELETAL
MUSCLE TISSUE ENGINEERING
Elowsson L (1,2), Kirsebom H (2), Carmignac V (1),
Durbeej M (1), Mattiasson B (2)
1. Muscle Biology Unit, Department of Experimental
Medical Science, Lund University, Sweden; 2. Department
of Biotechnology, Lund University, Sweden
Introduction. To use autologous materials for tissue
engineering would avoid problems with immune
reactions in vivo. We have developed a range of
macroporous scaffolds based on blood and plasma with a
cryogelation technique to be used for skeletal muscle
tissue engineering as an alternative treatment of injured
or diseased tissues. The most common treatments of
damaged skeletal muscular tissue are based on
autologous muscle transplantation and transposition,
however, these have shown a limited degree of success
[1-2].
Methods. Cryogelation of reaction mixtures based on
blood or plasma was carried out at -12°C where the
reaction took place. The structure and biomechanical
properties of the scaffolds were investigated. Myoblasts
were seeded on the scaffolds and cultured for 14 days.
The cultured myoblasts were evaluated by measuring cell
viability,
the
myogenic
phenotype
by
immunocytochemistry, and the cell morphology was
studied in electron microscopy.
Results and discussion. Both types of scaffolds had a
macroporous structure with interconnected pores. The
blood scaffold was found to have a higher elastic modulus
compared to the plasma scaffold, a lower swelling degree
and an uneven surface topography (Figure 1A). The
cultured myoblasts attached, migrated and proliferated
on both types of scaffolds. A typical myogenic
morphology was seen in scanning electron microscopy
(Figure 1B) and the immunocytochemistry confirmed a
myogenic phenotype (Figure 1C).
Conclusions. By using the patient’s own blood to create
macroporous scaffolds, either from whole blood or
plasma, together with the pre-culture of autologous cells
offers an easy, cost efficient and safe alternative for
successful tissue engineering. We are now investigated
these scaffolds in vivo.
(3.P1) DEVELOPMENT OF A NOVEL FIBRIN BINDING
PEPTIDE FOR INCORPORATION INTO BIOMATRICES
Rice J (1), Martino MM (1), Hubbell JA (1)
1. École Polytechnique Fédérale de Lausanne
Introduction. Engineered biofunctional scaffolds are
becoming an increasingly valuable tool for tissue
regeneration. For improved functionality the future
generation of biomatrices will need to incorporate
various morphogenetic compounds into the 3D matrix to
enhance the regenerative response and to encourage cell
migration within the injured site. Various methods have
been developed to integrate growth factors into the
matrix; some form covalent linkages to the biomatrix and
others rely upon affinity reagents incorporated within the
matrix. To expand upon the technology of integrating
growth factors within biomatrices, we used phage display
to identify a novel fibrinogen binding peptide for
increased retention in fibrin scaffolds.
Material and Methods. Peptide phage display was used
to identify a novel fibrinogen binding peptide. A
recombinantly produced fibronectin domain genetically
fused with the novel fibrinogen binding peptide was
produced. Fibrin gels were then formed in the presence
of the chimeric fibronectin protein and the release from
the gel was observed using ELISAs and western blots.
Results.The peptide has nanomolar affinity for fibrinogen
when displayed on the pIII protein of phage as
determined by ELISA. The peptide was then fused to a
growth factor binding domain of fibronectin and was
shown to increase the retention time of the fibronectin
domain by more than 10-fold, thereby allowing long-term
integration of growth factors into a fibrin matrix. The
protein/peptide fusion had mid-nanomolar affinity as
determined by biacore measurement.
Conclusion. In this work we identified a novel fibrinogen
binding peptide using phage display. Using this peptide
we created a variant of a fibronectin domain with the
ability to bind fibrinogen within a fibrin clot. This ability to
retain a therapeutic protein within a fibrin gel can be
used to improve the regenerative properties of fibrin
matrixes and enhance wound healing.
Keywords. fibrinogen, fibronection, biofunctionalization
(3.P2) ANALYSIS OF NEURONAL SPONTANEOUS ACTIVITY
IN VITRO: A MODEL TO ASSES THE EFFECT OF
IMPLANTABLE BIODEGRADABLE MATERIALS
Ortega J.A (1), Pérez M (1), Álvarez Z (2), Éngel E (2),
Álcantara S (1)
1. University of Barcelona; 2. Institute for BioEngineering
of Catalonia
Implantable biomaterials for CNS regeneration are
designed to be biodegradable and often to release
bioactive factors (BDNF, VEGF…). The regenerative ability
of these scaffolds might be modified by the presence of
by-products of degradation that can also have specific
bioactive properties. For instance neurons have a high
rate of oxidative metabolism and lactic acid is an
alternative energy source for them. Thus, lactic acid
produced by the degradation of Poly-Lactic Acid (PLA)
scaffolds might affect neuronal metabolism and
excitability. In this work we analyze the putative effect of
these factors by an in vitro approach that measures their
effect on the overall spontaneous activity of a neuronal
culture. E2dish technology is a novel method for
recording spontaneous neuronal electrical activity based
on microelectrode arrays. E2dish device uses pair of wells
connected by integrated micropipettes (microchannels).
The system allows the recording of the activity of neurons
whose axons sprout through the existing microchannel
between the pair of wells.
Here, we analyze the effect of lactate and BDNF on the
spontaneous electrical activity of a neuronal culture.
Preliminary results showed that lactate stimulates
spontaneous activity in vitro, probably by increasing the
formation of synapses in the neuronal cultures, as
indicated by increased synaptophysin protein levels in the
cultures. On the other hand, BDNF treatment dramatically
decreased the spontaneous activity in the neuronal
culture. BDNF treated cultures exhibited lower number of
burst, in particular lower fast repetitive firing activity.
BDNF raises synaptophysin protein levels, a marker for
synapses formation, as lactate does. Moreover it also
increased the levels of Calbindin, a GABAergic neuron
marker. Thus BDNF, in addition to increased
synaptogenesis, promotes the maturation of the
inhibitory system given us a plausible cause for the lower
spontaneous electrical activity in treated cortical
neuronal cultures.
Keywords. PLA, BDNF, electrophysiology, synapses
(3.P3) OSTEOBLAST ACTIVITY ON CARBONATED
HYDROXYAPATITE DISCS
Cartmell S (1), Rupani A (2), Hidalgo-Bastida LA (1), Dent A
(3), Turner I (3)
1. The University of Manchester; 2. The University of
Keele; 3. The University of Bath
Introduction. Hydroxyapatite (HA) is commonly used as a
bone substitute and as a scaffold for bone tissue
engineering. However HA has certain drawbacks such as
limited biodegradability and osteointegration properties.
This study investigates another form of HA, carbonated
hydroxyapatite (CHA), (which resembles the composition
of human bone), to potentially overcome these
drawbacks.
Materials and Methods. Experiments to assess the
potential of this novel scaffold. CHA discs (4.9 wt%
carbonate) in comparison to control HA discs were
carried out by seeding discs with MC3T3-E1 osteoblastic
cells. Analysis at 4 hours, 7days and 28 days included
SEM, Hydroxyproline assay (total collagen), Alamar Blue
assay, Live/Dead assay and realtime RT-PCR (collagen I,
collagen III and osteocalcin).
Results. Results indicate comparable cell adherence,
proliferation and viability of the osteoblast-like cells on
the CHA discs in comparison to HA discs. The SEM of the
CHA discs showed surface irregularities at 7 days
indicating dissolution (whereas the surface morphology of
HA remained consistent). Both CHA and HA discs showed
their surfaces to be covered by cells with evidence of
extracellular matrix production. The total collagen
production at 28 days, as evaluated by hydroxyproline
assay, did not show any statistically significant difference.
Real time PCR revealed an mRNA expression increase of
2.08, 7.62 and 9.86 fold for collagen I a1, collagen III a1
and osteocalcin respectively from cells seeded on the
CHA as compared to the HA discs (Figure 1).
Conclusion. In conclusion, the CHA was found to have
similar biological response to HA but also has the
potential to stimulate local osteoblastic cells to
upregulate bone related gene expression.
We would like to acknowledge Karen Walker (SEM, Keele
University), Sarah Rathbone (confocal microcopy, Keele
University) and Jaisal Patel (Bath University) and BBSRC
grant BB/F013892/1 for funding.
Keywords. carbonated hydroxyapatite, MC3T3-E1 cells,
proliferation, collagen production, gene expression
4. BIOINTERFACIAL ENGINEERING IN
REGENERATIVE MEDICINE
Chair: Antonio Peramo
Keynote speaker: Antonio Peramo
Organizer: Antonio Peramo
Synopsis: The interface between tissues and medical
implants is prone to infections and, over time, is not
conducive to the integration of the implant with the
tissue, ending with implant failure. These failures, with
higher rates for percutaneous implants due to the
permanent disruption of the skin, limit the time and
usefulness of the implants and cause significant health
care costs and patient morbidity. Seeking solutions for
these problems, the Symposium objective is to introduce
this area of research to the regenerative medicine and
tissue engineering communities. During the Symposium
we will discuss the problems associated with implants, in
a broad sense, and then the possible implementation of
regenerative techniques applied to the interfaces
between tissues and medical implants. Abstracts
describing
novel
technological
approaches
(ie
nanotechnology); implant surface modification; cell
delivery; tissue-implant integration (bone, skin or other
tissues); osseointegrated prosthesis; dental prosthetics;
and other research in the area of cell and tissue
engineering and biointerfacial engineering are welcome.
(4.KP) IMPLEMENTING REGENERATIVE MEDICINE AND
TISSUE ENGINEERING TECHNIQUES WITH SURGICAL
IMPLANTS
Peramo A (1)
1. University of Michigan
In this talk I will introduce and discuss the use of
regenerative techniques applied to the interfaces
between tissues and medical implants. This topic itself
represents a new area in regenerative medicine and has a
high potential to contribute to the current literature and
provide solutions in medical implants. The topic is highly
relevant to the theme 'Cells and Tissues as Advanced
Therapies' because cells and tissues will be used as
therapies around the implants. Among the topics for
discussion is the concept of dynamically introduce
regenerative materials and therapies at the tissueimplant interface. While this concept is valid for all
surgical implants, it will be most useful for devices that
are implanted for long periods of time or with higher risk
of failure, for instance, percutaneous devices.
Keywords. implant, percutaneous, bioengineering,
medical implant, biointerface
(4.O1)
IN
VIVO
ENDOTHELIALIZATION
OF
CARDIOVASCULAR
IMPLANTS
USING
DNAOLIGONUCLEOTIDES FOR ENHANCED CELL ADHESION
Schleicher M (1), Hansmann J (2), Bentsian E (2), Kluger PJ
(2), Liebscher S (1), Huber AJ (1), Fritze O (1), SchenkeLayland K (1), Schille C (2), Walles H (3), Wendel HP (4),
Stock UA (1)
1. Department Thoracic, Cardiac and Vascular Surgery,
University Hospital, Tübingen, Germany; 2. Fraunhofer
Institute for Interfacial Engineering and Biotechnology,
Stuttgart, Germany; 3. Department of Medical Materials
and Technology, UK Tübingen; 4. Department of
Congenital and Pediatric Cardiac Surgery, UK Tübingen
Current limitations of in vitro tissue engineering include
long in vitro culture, accompanied risk of infection and
cost intensive infrastructure. Accordingly this study
focuses on the development of concepts for in vivo
endothelialization. The objective of this study was
creation of cell adhesive DNA-oligonucleotide coatings on
heart valve and blood vessel surfaces. DNA is an intriguing
coating material with non-immunogenic characteristics
for easy and rapid chemical fabrication. For synthetic
surfaces a coating process with aminoparylene and
subsequent DNA-oligonucleotide adsorption was
established. In a second approach oligonucleotides were
covalently immobilized on decellularized bovine
pericardium
via
an
EDC
(1-ethyl-3-(3dimethylaminopropyl)
carbodiimide
hydrochloride)
mediated coupling. Immobilization of oligonucleotides
proved to be extremely stable. Coupled oligonucleotides
withstand shear stress up to 9,3 N/m2, which exceeds
physiologic shear stress conditions on heart valve and
vessel surfaces. Additionally incubation with human
serum up to 96 h showed no oligonucleotide degradation.
DNA-oligonucleotides enhanced endothelial cell adhesion
under continuous flow conditions significantly. The
oligonucleotide coating resulted in a more hydrophilic
surface, which facilitated protein adhesion from human
blood serum dilutions. This resulted in enhanced cell
adhesion. Biocompatibility was investigated by incubation
with human blood, granulocytes and thrombocytes and
by determination of released thrombogenic and
immunogenic factors. Immobilized oligonucleotides
revealed
low
thrombogenicity
and
good
hemocompatibility. Aminoparylene coated surfaces
showed no activation of thrombocytes, granulocytes, the
coagulation or complement system. Decellularized
pericardium however proved to be highly thrombogenic.
Crosslinking with EDC reduced the thrombogenic reaction
significantly. EDC-crosslinked tissue might open new
perspectives as matrix for in vivo tissue engineering.
Surface immobilization of oligonucleotides can facilitate
manufacturing of an “off-the-shelf” heart valve or blood
vessel for in vivo endothelialization. Additionally,
immobilization of oligonucleotides on other types of
implants where cell adhesion is desired opens new
opportunities for biocompatible coatings enhancing the
capability of incorporation in surrounding tissue.
Keywords. In vivo endothelialization, oligonucleotides,
hemocompatibility, cell adhesion
(4.O2) A FUNCTIONALLY GRADED SCAFFOLD THAT
MIMICS AN ORTHOPAEDIC INTERFACE AND CELLULAR
RESPONSE THEREOF
Samavedi S (1), Goldstein AS (1), Whittington AR (1)
1. Virginia Polytechnic Institute and State University
Introduction. A major concern with current scaffolding
strategies for the repair of anterior cruciate ligament
(ACL) injuries is poor osseointegration and subsequent
failure of the scaffold at the ligament-to-bone interface.
The natural ligament-to-bone interface consists of
gradients in mechanical and biochemical properties that
transition from soft unmineralized tissue to stiff
mineralized tissue. Therefore, we propose that a
functionally graded scaffold that mimics this transition
would possess suitable mechanical and chemical
properties to ensure spatially guided differentiation of
cells towards specific lineages.
Materials and Methods. In this study, a
polycaprolactone/nanohydroxyapatite (nHAP-PCL) blend
and a poly-(ester urethane) urea elastomer (PEUUR2000)
were co-electrospun from offset spinnerets to fabricate
graded scaffolds. Scaffolds were then treated with a 5x
simulated body fluid to superimpose a mineral gradient
atop the existing co-electrospun gradient. The presence
of gradients was demonstrated using dye assays and
microscopy. X-ray diffraction (XRD) and energy dispersive
spectroscopy (EDS) confirmed the presence of
hydroxyapatite on the surface of the nano-fibers.
Results. Mechanical testing indicated that the scaffolds
possess a gradient in tensile properties. In addition, the
failure mechanism of the graded scaffolds was elucidated
using real-time imaging in a micro-tensile tester. Finally,
MC3T3-E1 osteoprogenitor cells showed an up-regulation
of osteogenic markers in a graded fashion along the
length of the scaffold.
Conclusion. The study demonstrates that graded
scaffolds for orthopaedic applications can be fabricated
by employing appropriate polymers and suitable
processing techniques, and that these scaffolds can serve
as templates to study cell proliferation and
differentiation. Ongoing studies include the incorporation
of Bone Morphogenic Protein-2 into one set of
electrospun fibers in order to achieve a spatially graded
release of the protein and subsequent differentiation of
bone marrow stromal cells towards an osteoblastic
phenotype.
Keywords. Ligament-bone interface, Graded scaffold,
Electrospinning, Hydroxyapatite
(4.O3) TAILORING OF SURFACE PROPERTIES AT THE
NANOSCALE BY LAYER-BY-LAYER TECHNIQUE
Chiono V (1), Carmagnola I (1), Boccafoschi F (2), Gentile P
(1), Tonda-Turo C (2), Camacho Leal MDP (3), Ciardelli G
(2)
1. Dipartimento di Meccanica, Politecnico di Torino, Corso
Duca degli Abruzzi 24, 10129 Torino, Italy; 2.
Dipartimento di Medicina Clinica e Sperimentale, Facoltà
di Medicina, Università del Piemonte Orientale, 28100
Novara, Italy; 3. Dipartimento di Genetica, Biologia e
Biochimica, Centro di Biotecnologie Molecolari, Università
di Torino, Via Nizza 52, 10126 Torino, Italy
Introduction. A new generation of coronary stent systems
aimed at rapid re-endothelialization and able to protect
against thrombus formation and to minimize restenosis is
currently needed. The layer-by-layer (LbL) technique is a
versatile solvent-free processing allowing the coating of
surfaces with uniform ultrathin multilayered films to
tailor surface properties and structure at the nanoscale.
The aim of the work was the development of a LbL
coating with anti-thrombogenic properties and able to
support endothelization for vascular tissue engineering
and stent coating.
Materials and Methods. Stainless steel plates were
supplied by Carbostent Implantable Device (CID).
Aminolysed plates by 3-aminopropyltriethoxysilane
treatment (APTES; Sigma-Aldrich) were dipped in 0.1%
(w/v) heparin (HE; Sigma-Aldrich) aqueous solution for 15
min, subsequently rinsed with water, then dipped into a
0.1% (w/v) poly(diallyl dimethylammonium) chloride
(PDDA) aqueous solution for 15 min and dipped again in
water. Coatings with 1-11 layers were prepared. Surfaces
were characterised by contact angle analysis, FTIR-ATR,
SEM, AFM, XPS fluorescence microscopy, colorimetric
methods (UV-Vis). In vitro cell tests using endothelial cells
and haemocompatibility tests were also performed on
coated samples.
Results. Surface characterisation of stainless steel plates
after APTES treatment showed the presence of a
continuous coating of APTES containing amino groups for
further LbL coating. HE/PDDA coating was demonstrated
by FTIR-ATR analysis. LbL assembly of HE/PDDA was
shown by XPS analysis and a colorimetric method
(toluidine blue staining of HE). Endothelial cells were
found to attach and proliferate on LbL coated samples. HE
was found to contribute predominantly to the good
anticoagulation property of the HE/PDDA LbL coating.
Conclusion. A stable HE/PDDA LbL coating was developed
on stainless steel plates used as a model for metal stents:
the coating was able to promote re-endothelialisation
and showed improved anticoagulation properties.
NANOSTENT and ACTIVE projects are acknowledged.
Keywords. endothelization; layer-by-layer; stent; vascular
tissue engineering
(4.O4)
ADJUSTING
THE
ORIENTATION
OF
TROPOELASTIN: TARGETING CELL ADHESION TO SPECIFIC
POLYMER SURFACE LOCATIONS
Weiss AS (1), Bax DV (1), McKenzie DR (1), Bilek MMM (1)
1. University of Sydney
We recently described how human tropoelastin can direct
stem cell behavior (1). This protein is the soluble protein
precursor of elastin and has an integrin αvβ3 binding
motif at its C-terminal tip (2). The ability to generate cell
patterns on polymer surfaces is critical for the fabrication
of biosensors based on living cells, such as fibroblasts,
where it is necessary to monitor the status of these cells
in closely packed, defined locations (3). Accurate
positioning of cells is also a prerequisite for cell based
screening (4), cell separation techniques and for the
detailed study of cellular biology (5). Recent efforts to
pattern human cells on polymer surfaces have typically
used aligned microcontact printing, plasma mechanical
pattern generation (6), micro lithography, PDMS micropatterning and microfluidic patterning (7, 8) but these
methods are often associated with high cost, involve
complex surface chemistry and may not be applicable to
retain proteins in preferred orientations (5). There is a
paucity of ways to utilize intact ECM molecules to confer
biologically relevant cell interactions to the polymer
surface. Those methods that do rely on patterned
distribution of ECM proteins or protein-derived motifs on
a non-adhesive, often PEG-coated, background material;
this requires multiple complex chemical steps. We
present the use of surface plasma immersion ion
implantation polymer modification to both orient and
attach tropoelastin to enable the high resolution,
patterned distribution of human cells.
1. Holst J, et al. (2010) Substrate elasticity provides
mechanical signals for the expansion of hemopoietic stem
and progenitor cells. Nat Biotechnol 28(10):1123-1128.
2. Bax DV, Rodgers UR, Bilek MMM, & Weiss AS (2009)
Cell Adhesion to Tropoelastin Is Mediated via the Cterminal GRKRK Motif and Integrin alpha(V)beta(3).
Journal of Biological Chemistry 284(42):28616-28623.
3. Endler EE, Nealey PF, & Yin J (2005) Fidelity of
micropatterned cell cultures. Journal of Biomedical
Materials Research Part A 74A(1):92-103.
4. Khetani SR & Bhatia SN (2008) Microscale culture of
human liver cells for drug development. Nat Biotechnol
26(1):120-126.
5. El-Ali J, Sorger PK, & Jensen KF (2006) Cells on chips.
Nature 442(7101):403-411.
6. Ohl A & Schroder K (1999) Plasma-induced chemical
micropatterning for cell culturing applications: a brief
review. Surface & Coatings Technology 119:820-830.
7. Tan W & Desai TA (2003) Microfluidic patterning of
cells in extracellular matrix biopolymers: Effects of
channel size, cell type, and matrix composition on pattern
integrity. Tissue Engineering 9(2):255-267.
8. Nie Z & Kumacheva E (2008) Patterning surfaces with
functional polymers. Nat Mater 7(4):277-290.
Keywords. tropoelastin, elastin, plasma, interface
(4.O5) COPPER STIMULATES THE OSTEOGENIC
DIFFERENTIATION OF MESENCHYMAL STEM CELLS
Burghardt I (1), Lüthen F (1), Prinz C (2), Neumann HG (2),
Rychly J (1)
1. Laboratory of Cell Biology, Medical Faculty, University
of Rostock; 2. DOT GmbH
Introduction. In context with the design of medical
implants which both stimulate the regeneration of bone
tissue and are suitable to prevent infection due to
bacteria, we have been interested in the effects of copper
ions on the osteogenic differentiation of human
mesenchymal stem cells (MSC). We hypothesized that the
release of copper from an implant surface induces
bacterial death. However, because of the known
physiological role of copper, lower concentration in a
later phase after implant incorporation or at greater
distances from the implant surface could have a
stimulating effect on stem cells.
Materials and Methods. Mineralization of cells as a
marker for osteogenic differentiation was measured by
calcein bound to extracellular calcium phosphate and
visualized by laser scanning microscopy.
Results. The critical concentration of copper ions for the
survival of MSC was 0.5 mM. Therefore we studied the
effect of copper on the osteogenic differentiation below
this concentration. We found that when adding CuSO4
into a medium for osteogenic differentiation, copper ions
stimulated the osteogenic differentiation of adherent
cells with a maximum at a concentration of 0.3 mM.
Copper induced a stronger mineralization when the cells
were cultured on cell culture polystyrene than on
titanium oxide or titanium surfaces. To see, whether
copper implemented into implant materials induces an
osteogenic differentiation of MSC, cells were cultured on
calcium phosphate surfaces containing copper salts.
These surfaces enhanced the osteogenic differentiation of
adherent cells compared with copper free surfaces.
Concerning possible mechanisms which are involved in
the biological response induced by copper, we revealed
that copper affected the strength of cell adhesion and the
expression of various integrins.
Conclusion. Copper containing implants are suitable to
promote bone regeneration by the stimulating effect on
the osteogenic differentiation of mesenchymal stem cells.
The work was supported by the government of
Mecklenburg-Vorpommern
(V230-630-08-TFMV-S016/F016).
Keywords. copper, implant, mesenchymal stem cell
(4.O6) A NEW GDF-5 MUTANT MEDIATING SUPERIOR
TABECULAR AND CORTICAL BONE FORMATION IN A
CRITICAL SIZE DEFECT RABBIT MODELL
Holschbach J (1), Kleinschmidt K (2), Plöger F (3),
Glockenmeier J (4), Kretzer JP (1), Richter W (1)
1. Research Center for Experimental Orthopaedics,
Orthopaedic University Hospital Heidelberg; 2.Merck
KGaA, Darmstadt; 3. Biopharm GmbH, Headquarter
Heidelberg, Heidelberg; 4. University Hospital Heidelberg,
Department for Orthopaedic, Trauma Surgery and
Paraplegiology, Spinal Cord Injury Center
Treatment of large bone defects remains a challenge for
orthopaedic surgeons. In clinical use for this indication
are Bone morphogenetic proteins (BMPs) which are
potent agents to induce bone formation. The
osteoinductivity of human growth-and-differentiationfactor-5 (GDF-5) is well established, but a reduced
amount of ectopic bone is formed compared to other
members of the BMP-family like BMP-2. We found
previously, that swapping two amino acids in GDF-5 to
residues contained in BMP-2 increased osteogenicity of
emerging GDF5V453/V456 (mt) and enhanced its ectopic
bone formation capacity compared to wildtype GDF-5.
Aim of this study was to investigate the potency of GDF5mt for treatment of critical size bone defect (CSD) in
comparison to BMP-2 and GDF-5.
Bone formation in CSD in rabbit radii treated with BMP-2,
GDF-5, GDF-5mt or buffer solution was assessed by in
vivo µCT scans at 4, 8 and 12 weeks post surgery.
All GDF-5mt treated defects bridged after 4 weeks, while
only 6 of 9 BMP-2 treated bones were bridged at 8 weeks.
After 12 weeks GDF-5mt increased bone volume
compared to BMP-2 and GDF-5 treated animals
(p<0.001). Bone marrow cavities were remodelled in all
GDF-5mt treated animals during 8 weeks, while BMP-2
mediated callus remained spongy at 12 weeks post
surgery. Micro morphological parameters in BMP-2, GDF5 and control defects differed significantly from the GDF5mt group as well as from contralateral healthy bone.
Concomitantly, micro architectural parameters were
similar in the GDF-5mt group and healthy bone. GDF-5
wildtype mediated cartilaginous gap formation in 5 of 9
animals - an effect that was not detectable after BMP-2
or GDF-5mt teatment after 8 weeks.
The GDF-5mt showed superior bone formation capacity
than GDF-5, and a faster induction and cortical bone
formation than BMP-2. GDF-5mt thus represents a
promising new growth factor variant promising improved
outcome in bone regeneration strategies.
Keywords. Growth factors, GDF-5, BMP-2, GDF-5mt,
Rabbit, Bone healing
(4.O7) BIODEGRADABLE DISULFIDE-CATIONIC POLYMER
FOR THE GENE THERAPY OF RECESSIVE DYSTROPHIC
EPIDERMOLYSIS BULLOSA
Aied A (1), Cao H (1), Dong Y (1), Zheng Y (1), Pandit A (1),
Wang W (1)
1. NUI, Galway
Introduction. Dystrophic epidemolysis bullosa (DEB) is a
group of inherited diseases characterized by the blistering
and scarring of the skin after mild trauma. The most
(4.O8)
MULTI-SCALE,
HIERARCHICALLY
PLLA/POROUS TITANIUM HYBRIDS FOR
REPLACEMENT
POROUS
TRACHEA
Vrana NE (1), Dupret-Bories A (1,3), Chaubaroux C (1),
Schultz P (3), Debry C (1,3), Coraux C (4), Vautier D (1,2),
Lavalle P (1,2)
1. Institut National de la Santé et de la Recherche
Médicale, INSERM Unité 977, France; 2. Faculté de
Chirurgie Dentaire, Université Louis Pasteur, France ; 3.
Hôpitaux Universitaires de Strasbourg, France ; 4. Institut
National de la Santé et de la Recherche Médicale, INSERM
Unité 903, France
In trachea regeneration, the two most persistent
problems are restenosis of the tracheal lumen by
migration of cells and lack of epithelialization. For this
end, we developed a hierarchically porous (from
macropores to nanopores) PLLA/ porous Titanium hybrid
scaffold, that can prevent restenosis by fibroblastic cells
by size exclusion and that can promote epithelialization
by a surface of either nanofibrillar or nanoporous nature
(Nanoporous PLLA films or Collagen/Alginate fibrillar
polyelectrolyte multilayers). Moreover, since the
necessary volume for tissue regeneration would be lower
due to the presence of the titanium body, vascularization
of epithelium layer would occur faster. This hypothesis
was tested invitro by quantifying fibroblast migration
through the scaffold and via human respiratory cell
culture. Fibroblast movement was significantly impeded
by the microporosity gradient and a confluent layer of
epithelial cells was obtained.
The hypothesis was further tested in a rabbit model (New
Zealand white rabbits) with a 2cm length full
reconstruction model with implantation duration of 6
weeks. CRP levels of animals were checked regularly also
after removal of the implants and implants were
characterized
for
fibroblast
movement
and
epithelialization, infection and polymer degradation.
Results showed that, the porosity gradient effectively
prevented clogging of the lumen by the migrating cells
and the top film layer was in place after 4 weeks.
Epithelial migration was evident but incomplete. Polymer
degradation was most prominent at the outer surface
were most of the remodeling took place.Fibrovascular
tissue development within the pore structures was
apparent. For a more in-depth understanding, cytokine
composition of the blood of the implanted animals is
being investigated now. Our results suggest that, the
developed hybrid scaffold can successfully replace
tracheal segments. However, for long segments, preepithelialization with patients own respiratory epithelium
is necessary as the rate of migration is not good enough
for full coverage.
Keywords. In-vivo, Trachea, Titanium, Pore gradient, PLLA
From micro to nano porosity
severe case being transmitted by the autosomal recessive
pattern and is known as recessive dystrophic
epidermolysis bullosa (RDEB). The overall aim of the
project is to demonstrate direct gene delivery of COL7A1
plasmid carrying the correct COL7A1 sequence to human
RDEB skin cells using a biodegradable cationic polymer
(termed DMA) and a thermoresponsive hydrogel.
Materials and Methods. Polymer synthesis: The polymer
was synthesised by deactivation enhanced atom transfer
radical polymerisation (DE-ATRP) at 60
˚C for 6 hours
under argon and then characterised by gel permeation
chromatography (GPC) and proton nuclear magnetic
resonance (NMR). Transfection and cell viability studies:
Mouse 3T3 fibroblasts (DMEM, 10% FBS and 1%
penicillin/streptomycin) (Sigma Aldrich) and Human
primary keratinocytes from RDEB patients (keratinocyte
growth medium II, supplement mix and CaCl2)
(Promocell) were transfected with DMA/DNA at optimal
weight to weight ratios (w/w). Alamar Blue™ (Invitrogen)
was used to analyse the cellular metabolic activity.
Indirect immunofluorscence: COL7A1 protein expression
from RDEB primary human keratinocytes was visualised
using polyclonal rabbit primary antibody to human
COL7A1 protein and Alexa Flour® goat anti-rabbit
secondary antibody. DAPI was used to stain the nucleus.
Results. The polymer showed higher transfection
efficiency while maintaining high cell metabolic activity
compared to superFect® and LipofectamineTM. Cells that
were treated with DMA/COL7A1 polyplexes showed
typical patterns of expression of collagen VII (COL7A1)
protein compared to untreated cells.
Conclusion. The results suggest direct and long lasting
treatment of RDEB using a biodegradable polymeric gene
vector has a potential therapeutic application.
DEBRA Ireland and Austria, Heath Research Board (HRB)
of Ireland (HRA/2009/121), Science Foundation Ireland
(SFI) Principal Investigator and Stokes Lectureship
Programmes (10/IN.1/B2981 and 07/EN/E015A), and
National University of Ireland, Galway (Scholarship).
Keywords. Gene Therapy
Z section of Titanium-polymer material
(4.P1) CELL ADHESION PROMOTING RGD-SILK
Nilsson AY (1), Meinel AJ (1), Panke S (1)
1. ETH Zurich, Switzerland
Introduction. Silk, a biocompatible and biodegradable
material with good mechanical properties, is a suitable
scaffold material for e.g. bone tissue engineering. By
introducing the cell signaling amino acid sequence RGD
directly into the primary sequence of a genetically
engineered silk we hypothesize that receptor-mediated
cell adhesion and spreading will be promoted.
Materials and Methods. A DNA construct coding for silk
based on the major ampullate spidroin 1 from the spider
Nephila clavipes has previously been assembled (15mer)
[1]. We have added DNA coding for the fibronectin
derived amino acid sequence VTGRGDSPA both up and
downstream of the 15mer gene to create RGD-15mer.
The two engineered silks were produced by fed-batch
fermentation using a bacterial expression system, purified
and cast into films. The films were seeded with DiI stained
human mesenchymal stem cells (hMSCs), and cell
adhesion was studied with time-lapse microscopy.
Results. hMSCs seeded on the RGD-15mer silk started to
polarize and migrate, something that could not be
observed on the 15mer silk over the duration of the timelapse study (2.5 hours). The experiments were performed
with serum free medium. Currently osteogenic
differentiation on the different materials is being studied.
Conclusions. We have set up a production system for
engineered silk materials which we could utilize to
produce a silk with integrated RGD sequences. This
material showed improved cell adhesion and has the
potential to be used as a scaffold material for tissue
engineering purposes.
References. [1] Bini E, Foo CW, Huang J, Karageorgiou V,
Kitchel B, Kaplan DL (2006) Biomacromolecules 7:313945.
We thank Professor David Kaplan (Tufts University,
Medford, MA) for kindly providing the Nephila clavipes
silk (15mer) gene containing plasmid, and Dr. Kristopher
Kubow (ETH Zurich) for assistance with the time-lapse
studies. This work was supported by the BioEngineering
Cluster (ETH Zurich).
Keywords. Silk, RGD, mesenchymal stem cells
(4.P2) NORMAL HUMAN OSTEOBLASTS RESPONSE TO
PECVD TIO2 FUNCTIONALIZED PLGA MEMBRANES
DESIGNED FOR GUIDED TISSUE REGENERATION (GTR)
Salido M (1), Terriza A (2), Vilches JI (3), Díaz-Cuenca MA
(2), Barranco A (2), González-Elipe AR (2), Vilches J (3)
1. School of Medicine. University of Cádiz; 2. Instituto de
Ciencia de Materiales. Seville (CSIC-Univ. Seville); 3. School
of Dentistry. University of Seville. Spain; 4. School of
Medicine. University of Cádiz
Introduction. The therapeutical approach for reparation
of bone defects at the maxillofacial level is now focused
to bony tissue reparation, and minimization of connective
healing and the recovery time. Guided tissue
regeneration (GTR) specifically aims to overcome some
limitations of conventional therapy. Aliphatic polyesters polyglycolic acid, polylactic acid and their bioresorbable
copolymer (PLGA)- are arousing a great interest and are
approved for the US FDA for certain human clinical use.
They can be employed as supporting or stabilizing
elements for bioactive materials, i.e.titanium, and their
degradation products are removed by natural metabolic
pathways. Porous three-dimensional temporary scaffolds
play an important role in manipulating cell function and
guidance of new organ formation, and their surface
chemical composition is a key factor for achieving a
durable osseointegration. The establishment, through an
“in vitro” study, of the osteoinductive (GTR and
mechanotransduction) properties of TiO2 PECVD
functionalized and non functionalized PLGA membranes
on human osteoblasts.
Material and Methods. Human osteoblasts were grown
on TiO2 functionalized and non functionalized PLGA
membranes produced in the ICMSE, Seville, Spain.
Rhodamine-phalloidine and antivinculin immunolabelled
labelled cells were analyzed after 24 and 48 h in culture.
Results. Osteoblasts grown on non functionalized PLGA
shown an elongated shape, and distributed in a fascicular
pattern, similar to those growing on glass, with small focal
contacts all along the cell body (A,B). Osteoblasts cultured
on TiO2 PECVD functionalized PLGA surfaces grown and
polarized into an organized reticular pattern, with well
developed stress fibers oriented to gross focal adhesion
points (C,D).
Conclusion. Our results demonstrate that PECVD TiO2
functionalization of PLGA surface induces osteoblasts
organization into a reticular pattern that could be more
efficient for bone formation in those locations, like
maxillofacial bone, that support non oriented and
complex mechanical loadings.
Keywords. osteoblasts, guided tissue regeneration, TiO2,
functionalization, osseointegration
(4.P3)
MEASUREMENTS
OF
POLY
NISOPROPYLACRYLAMIDE-CO-BUTYLACRYLATE/3T3 CELLS
INTERACTIONS BY ATOMIC FORCE MICROSCOPY
Becerra N (1), Andrade H (2), López B (1), Restrepo L (1),
Raiteri R (2)
1. Univesidad de Antioquia; 2. Università degli Studi di
Genova
Poly N-isopropylacrylamide-based (P(NIPAAm) ) hydrogels
has already been proposed as cell culture support for cell
sheet engineering because its thermosensibility
associated with a lower critical solution temperature
(LCST around 32°C). The hydrophobic/hydrophilic
character of P(NIPAAm) hydrogels allows cell growth
above LCST and cell release below it, respectively.
We have observed that poly N-isopropylacrylamide-cobutylacrylate copolymer ( P(NIPAAm-co-BA) ) is more
hydrophobic than homopolymer P(NIPAAm). This
characteristic is known to improve cell adhesion,
increasing cell-hydrogel interactions through an efficient
adhesive proteins adsorption such as fibronectin on the
hydrogels. Therefore we propose the use of P(NIPAAmco-BA) as cell culture support in cell sheet engineering
once
an
adequate
balance
between
hydrophobic/hydrophilic character is provided.
In this work an Atomic Force Microscope (AFM, model5500, Agilent Technologies) was used to characterize cellthermosensitive hydrogels interactions at two different
temperatures, above and below copolymer LCST.
Polystyrene (PS) microbeads was glued to a
microcantilever and coated with P(NIPAAm-co-BA)
copolymer using a micro-manipulator. Uncoated PS
microbeads were the controls. 3T3 Swiss cells were
cultured, 24 hours after passage were used in AFM
experiments. Maps of force versus distance curves (8 x 8
curves) at 37°C and 25°C were recorded. For each curve
(Figure) the PS microbead was brought and kept into
contact with a single cell for 10 seconds, afterwards the
cantilever was withdrawn and force necessary to
microbead detachment from cell was measured. Data
were acquired and analyzed using a software developed
in LabVIEW (National Instruments, Austin TX).
Maximum adhesion distributions obtained at 25°C and
37°C show a higher adhesion force above the LCST of the
P(NIPAAm-co-BA) copolymer, which confirms the
dependence of cell-hydrogel interactions with
temperature and the possibility of cell release at 25°C.
These results support the use of P(NIPAAm-co-BA)
copolymer as a cell culture substrate in cell sheet
engineering.
Convocatoria Fac-Medicina, Sostenibilidad 2009-2011,
Colciencias-doctorados Nacionales-2008
Keywords.
cell
sheet
engineering,
Poly
Nisopropylacrylamide, Atomic Force Microscope
(4.P4) DEVELOPMENT OF AN IN VITRO 3D MODEL TO
SIMULATE THE HUMAN BLOOD-CEREBROSPINAL FLUID
(B-CSF) BARRIER
Appelt A (1), Taichrib K (1), Schubert-Unkmeir A (2),
Slanina H (2), Walles H (1)
1. Chair Tissue Engineering & Regenerative Medicine,
University Clinic Würzburg; 2. Institute of Hygiene and
Microbiology, University Würzburg
Introduction. Neisseria meningitidis is a strictly humanspecific pathogen with the capacity to cause septic shock
and meningitids. Therefore, the aim of this study was to
construct an endothelial cell barrier in order to develop
an in vitro 3D model of a human B-CSF barrier using
human brain microvascular endothelial cells (HBMEC).
The subarachnoideal space was constituted by a
biological vascularized scaffold of collagen I/III (BioVaSc).
Culture was performed in static and dynamic conditions.
Materials and Methods. The BioVaSc was processed from
porcine jejunum by mechanical, chemical and enzymatic
decellularization. Different cell concentrations and
culture periods were tested under static conditions.
Additionally, a dynamic culture was performed mimicking
the bloodstream. The confluence of the endothelial
monolayer
was
verified
by
measuring
the
transendothelial electrical resistance (TEER). The static
and dynamic studies were also repeated with primary
microvascular endothelial cells isolated from human skin.
Both cell types were characterized with histological
staining against the cluster of differentiation molecule 31
(CD31) and the von-Willebrand-Factor (vWF).
Results. The static culture tests of HBMEC’s on the
BioVaSc revealed an optimal cell concentration of 2x10^5
cells/cm² BioVaSc and an optimal cultivation period of 2-5
days. With these conditions a cell monolayer was
established. However, the monolayer wasn’t tight and the
cells often grew in untypically multiple layers. In contrast
the primary endothelial cells formed a tight monolayer
under static conditions. Dynamic culture conditions in a
flow chamber resulted in the formation of a tight
monolayer of HBMEC’s, confirmed by TEERmeasurement. The histological staining exposed that the
cell line HBMEC in contrast of the primary endothelial
cells had lost the endothelial markers CD31 and vWF.
Conclusion. The results of our study show the
construction of a tight endothelial cell barrier under
dynamic culture conditions. The next steps should be to
complete the B-CSF models with meningeoma cells and to
infect them with Neisseria meningitidis.
Keywords. blood-cerebrosoinal fluid barrier, in vitro 3D
model, collagen I and III scaffold, tight HBMEC monolayer
(4.P5) NANOLAYERS OF PEVCD TIO2 SUITABILITY FOR
HUMAN OSTEOBLASTS GROWTH FOR TISSUE
ENGINEERING
Salido M (1), Terriza A (2), Torres D (3), de la Orden E (1),
Barranco A (2), Díaz-Cuenca MA (2), Vilches J (1),
González-Elipe AR (2)
1. School of Medicine. University of Cádiz; 2. Instituto de
Ciencia de Materiales. Seville (CSIC-Univ. Seville); 3. School
of Dentistry. University of Seville, Spain
Introduction. Bone regeneration can be enhanced
through implantation of biocompatible scaffolds. The
complexity of scaffolds surfaces could positively influence
osteoblastic mechanotransduction. Surface chemistry
plays an important role in implant fixation and can
directly influence osteoblasts adherence, attachment,
spreading and metabolism modifying and controlling the
osseointegration process. The use of an appropriate
template to provide physical support and a local
environment is essential for a successful regeneration.
With the aim of tailoring suitables surfaces to be tested in
vitro, scaffolds activation by Plasma enhanced chemical
vapour deposition of Ti appears as an alternative to wet
chemical treatments
Material and Methods. Human normal osteoblasts were
grown on PEVCD TiO2 functionalized and non
functionalized TiO2 PET samples, produced in the ICMSE
(Instituto de Ciencias de Materiales) in Seville, Spain.
Rhodamine-phalloidine and antivinculin fluorescent
labelled cells were analyzed after 24 and 48 h in culture.
Results. After living osteoblasts examination (phase
contrast and DIC microscopy), phenotypical cell changes,
like filopodial and lamellopodial emission, mainly
oriented to elongation, alignment and focal adhesions
towards the growing surface were observed. Actin
cytoskeleton immunolabelling of growing cells revealed a
higher polarization and stress fiber development,
together with a more defined osteoblast orientation
induced by surface, in cells grown on the 100 nm PEVCD
TiO2 functionalized PET samples.(Figure: actin
immunolabelling of osteoblasts grown on A: glass; B: PET;
C,D:PEVCD TiO2 PET)
Conclusion. Surface chemistry of the scaffolds plays a key
role in osseointegration. In order to render a nanolayer
with controlled structure and composition to enhance
osteoblasts adherence and differentiation, TiO2 thin films
prepared by plasma enhanced chemical vapour
deposition were grown on PET samples.Our present
results demonstrate the suitability of PEVCD as an
alternative for surface functionalization of polymers that
can lead to the development and tailoring of new
bioreabsorbable polymeric membranes for bone tissue
regeneration.
Keywords. TiO2 nanolayers, osteoblasts, bone
regeneration, osseointegration
5. BIOMATERIALS & ENGINEERED
CONSTRUCTS-OUTCOMES IN
MEDICINE/EXISTENT SURGERY
(BECOMES)
Chair: Amulya K. Saxena
Co-chairs: Richard Ackbar, Herwig Ainoedhofer
Keynote speaker: Amulya K. Saxena
Organizer: Amulya K. Saxena
Synopsis: Biomaterials development resulting from
extensive basic research has to be translated in the
clinical setting to determine their suitability or their
shortcomings in human applications.
Translational
research will involve the investigation of biomaterials that
have been developed under optimal laboratory
conditions, but have to be utilized under complex clinical
and surgical pathological states. The Biomaterials &
Engineered Constructs- Outcomes in Medicine/ Existent
Surgery (BECOMES) Group focuses on the translation
outcomes of biomaterials and generated tissues in clinical
and contemporary surgical applications.
The group focuses also on the better understanding of
the clinical pathology and relating the difficulties
experienced by the clinicians and surgeons in their
practise to the tissue engineering community. The group
aims to highlight to Researchers in Tissue Engineering the
coexistence of conditions (co-morbidities) that will affect
or alter the primarily intended functioning of the original
biomaterial or engineered tissue. The foremost intention
of the group is to expose the Basic Science Research
Community in Tissue Engineering with the ground
realities in patient pathology and the difficulties
experienced by the Medical Practitioners and Surgeons.
This exposure is intended to help in the translational
research and evaluation and implementation of Tissue
Engineering Technologies in Clinical practice.
The symposia intention of the BECOMES Group is 4 fold:
1. Outcomes of Biomaterials in Contemporary Clinical
Applications: Presentations will be invited from
researchers who have applied biomaterials or engineered
constructs in the clinical practise. These presentations
are intended to showcase the ease or difficulties in the
application of these materials in humans. The usage of
these materials, their outcomes and their shortcomings
will be presented and technical improvements that are
desired will be presented to researchers in the area of
Tissue Engineering.
2. Identification of Clinical states demanding
Regenerative Medicine: The second area of presentation
will be exploring the clinical states that require
biomaterials or engineered tissues. Presentations will be
made to expose clinical conditions and the present state
of palliative therapies that are offered to the patients.
These presentations are intended to expose tissue and
clinical states that have not part of the frontline research
in tissue engineering, however the demand of tissue is
these area is so dire that millions of Euros are being spent
in the management of these patients with no optimal
solutions in sight.
3. Focus on Paediatric organ loss: The focus of the tissue
engineering research is mainly on the adult populations
and the conditions encountered later in life. There is
even a much larger shortage of organs in the newborns,
infants and the childhood age group that the tissue
engineering research community is not aware about.
Paediatric organ shortages are further complicated by
donor mismatches (for example if an adult liver donor is
found for a newborn who requires a liver transplant- it is
almost impossible to fit an adult liver in the child).
Biomaterial research in in-vivo animal models:
Presentations will be also done on in-vivo animal models
to explain the working of biomaterials or generated
tissues in these experimental studies.
These
presentations will be important for the clinicians and
surgeons to understand the development and the present
stage of research in animal experiments and the future
clinical applications.
(5.KP) TISSUE ENGINEERING FOR CLINICAL SYNDROMES:
EXPECT THE UNEXPECTED MICROENVIRONMENT
Saxena AK (1)
1. Experimental Fetal Surgery & Tissue Engineering Unit,
Department of Pediatric- & Adolescent Surgery, Medical
University of Graz, Austria
Clinical syndromes which affect multiple organs are
indirectly a target of research for the Tissue Engineering
& Regenerative Medicine groups worldwide. However,
while the present research focuses on the development
of single organs by interest groups; syndromes that affect
multiple organs, present a multimorbid patient, the
affection of which changes the microenvironment for the
transplanted tissue engineered organ. At present organ
development is focused on application of strict protocols
for cell isolation, seeding on scaffolds under the “near
perfect” conditions to generate in-vitro, in-situ and in-vivo
neotissues. Such neotissues are then envisaged for
implantation or transplantation in an individual that offer
near to physiological conditions for the neotissue to
assimilate, incorporate and integrate. Clinical syndromes
affecting major organs soft tissue organs such as the
heart, lungs, liver, kidneys and intestine are a focus of this
presentation to better demonstrate the ground realities
faced in the starting of trials of tissue engineering organs.
The impact of these tissues in syndromes is so large that
normal tissues within the body are forced to alter their
function and structure in such individuals.
Major clinical syndromes can further be divided for better
understanding under those affecting the pediatric
population to those that can further continue to affect
the individual later as an adult. These can be further
divided into those affecting the clinical status of the
patient versus those that influence states that necessitate
surgical corrections. It is important for the Tissue
Engineering & Regenerative Medicine community to be
aware of these imperfect microenvironments and at
some stage work on the ground realities that will
determine the success or failures of neotissue implants
and transplants. Future work in Tissue Engineering &
Regenerative Medicine should focus on these altered
microenvironments for successful implementation of this
technology in the clinical and surgical patient.
(5.O1) NOVEL BIODEGRADABLE VASCULAR PROSTHESIS:
SHORT-TERM RESULTS AFTER CAROTID ARTERY
REPLACEMENT IN THE PIG
Walpoth BH (1), Mrowczynski W (1), Mugnai D (1), de
Valence S (1), Tille JC (1), Khabiri E (1), Gurny R (1),
Kalangos A (1), Moeller M (1)
1. Geneva University Hospital
Introduction. There is a continuous search for synthetic,
shelf-ready, coronary artery bypass grafts. Biodegradable
scaffolds, repopulated by recipient’s cells regenerating a
neo-vessel, can be a suitable option for both adult and
pediatric, urgent and elective cardiovascular procedures.
We assessed a new biodegradable vascular prosthesis for
arterial replacement in the pig.
Materials and Methods. Ten anesthetized pigs
underwent bilateral carotid artery replacement with
biodegradable electrospun Poly(ε-caprolactone) (PCL)
nanofibre prostheses (4mm-ID; 5cm-long); or expandedpolytetrafluoroethylene (ePTFE) prostheses serving as
control. Peri-operative anticoagulation was achieved with
intravenous heparin (double baseline ACT). Postoperatively, until conclusion of the study at 1-month,
animals received aspirin daily. Transit
Time Flow (TTF) was measured intra-operatively and at
sacrifice. Doppler ultrasound follow-up was performed at
1 and 4 weeks when a selective carotid angiography
assessed patency. Graft examination consisted of
histology with special stainings, planimetry and SEM.
Results. Surgical handling and haemostasis of the new
prostheses were excellent. Patency rate was 78% (7/9) for
PCL grafts, compared to 70% (7/10) for ePTFE grafts. TTF
and Doppler ultrasound showed no significant changes in
flow and velocity or diameter over time in both groups.
Both prostheses showed minimal in vivo compliance as
compared to native carotid artery. Neoendothelialisation
was 79% for PCL (Fig.1:A,B) and 80% for ePTFE grafts.
Neointima formation was limited in both grafts. The PCL
graft was partially infiltrated from the adventitia by
macrophages, myofibroblasts and capillaries with a mild
foreign-body reaction and focal thrombus formation
(Fig.1:C).
Conclusion. Biodegradable, electrospun PCL grafts
showed good surgical properties, no aneurysm formation
and similar short-term patency compared to ePTFE grafts.
Rapid, good endothelialisation and cell ingrowth confirms
the hypothesis of in vivo vascular tissue engineering.
Despite good early results long-term follow-up is required
before clinical application such as CABG.
Keywords. tissue engineering, scaffolds, animal
experiments
(5.O3) ROLE OF SIDE POPULATION CELLS DURING
WOUND HEALING IN RAT VOCAL FOLDS
Gugatschka M (1), Kojima T (2), Ohno S (2), Kanemaru SI
(2), Hirano S (2)
1. ENT University Hospital Graz, Medical University Graz,
Austria; 2. Department of Otolaryngology- Head & Neck
Surgery, Graduate School of Medicine, Kyoto University,
Kyoto, Japan
Introduction. Despite big advances in understanding
mechanisms of wound healing in vocal fold injury, it still
remains unclear which are the decisive factors that lead
to a complete restoration or to scarring. Among several
other factors, stem cells are believed to play an important
role in vocal fold restoration. Side population (SP) cells
are considered to contain high numbers of stem cells and
have gained great interest in the tissue engineering
community. Aim of the following study was to investigate
the recruitment pattern of SP cells in a rat vocal fold
injury model.
Materials and methods. Unilateral vocal fold scarring was
performed in Sprague Dawley rats. Larynges were
harvested 1, 3, 5, 7, 14, 21 and 35 days after initial injury
and examined immunohistochemically for the presence
of SP cells. This was done in coronal sections of the
posterior and anterior macula flava as well as in the midportion of the vocal fold investigating the lamina propria.
Results. Number of SP cells peaked significantly after 7
days in the mid-portion of injured vocal folds, with a
return to pre-injury levels after 14 days. No increase was
detected throughout the observed time in the contra-
lateral side. Number of SP cells increased slightly but not
significantly in both anterior and posterior macula flava.
Conclusion. Our findings suggest that SP cells may play an
important role in early vocal fold wound healing and may
serve as a possible therapeutic target.
Keywords. Side population cells - wound healing vocal
folds
(5.O4) NEW POLYMER COATING TO VISUALIZE SURGICAL
MESH BY MRI
Guillaume O (1), Blanquer S (1), Letouzey V (1), Lemaire L
(2), de Tayrac R (3), Garric X (1), Coudane J (1)
1. IBMM, Artificial Biopolymers Group, UMR-CNRS 5247,
UM1-UM2, 15 Av. C. Flahault, 34093 Montpellier, France;
2. INSERM UMR-S 646, Angers University, 10 rue André
Boquel, 49100 Angers, France; 3. Department of
Obstetrics and Gynecology, Carémeau Hospital, 30000
Nîmes, France
Introduction. Magnetic Resonance Imaging (MRI) is
widely used for both clinical diagnosis and/or staging of
human diseases [1]. Unfortunately, MRI still is a
powerless imaging technique for prostheses observation
post-operatively [2]. To circumvent this drawback, we
synthesised a new MRI visible polymer by grafting a MRI
contrast agent on the polymer backbone. Its potential for
clinical use was evaluated using in vitro and in vivo
experiments.
Materials and Methods. Anionic activation of
poly(methyl acrylate) (PMA) chain was performed using
lithium diisopropylamide (LDA) [3]. The resulting
macropolycarbanion was then reacted with a chelate of a
MR contrast agent based on Gadolinium (DTPA-Gd). The
in vitro polymer cytotoxicity was investigated using L929
fibroblasts by cells viability and pro-inflammatory
response assays. The PMA-DTPA-Gd polymer was coated
on commercial polypropylene meshes by spray coating. In
vitro MR images were performed on coated meshes
embedded in agarose gel. For in vivo visualization, coated
meshes were implanted in a Wistar rat’s back and MR
images were obtained 10 days after implantation using an
experimental (7 T) and a clinical (1.5 T) MR apparatus.
Results. MR contrast agent (DTPA-Gd) has been
covalently grafted on poly(methyl acrylate) and in vitro
cell investigations of the grafted polymer revealed good
in vitro cytocompatibility associated to a limited toxicity.
After coating, this new polymer allowed to significantly
enhance in vitro MR signal of the meshes for a long-term
period. After implantation in rat, the coated meshes were
unambiguously detectable whatever the location and the
morphology was clearly recognizable.
Conclusion. To our knowledge, it is the first nonhydrosoluble MRI visible polymer ever described. This
polymer, once coated on an initial MR transparent
polypropylene mesh, induces in vivo MR signal
enhancement for a long time period and allows a quick
MRI localization of the device.
References.
1. Yan, G.-P., Magnetic resonance imaging contrast
agents, an overview. 2006.
2. Boukerrou, M., et al., [MRI evaluation of surgical pelvic
floor repair]. Gynecol Obstet Fertil, 2006. 34(11): p. 10248.
3. Ponsart, S., J. Coudane, and M. Vert, A novel route to
poly(epsilon-caprolactone)-based copolymers via anionic
derivatization. Biomacromolecules, 2000. 1(2): p. 275-81.
(5.P2)
PREPARATION
OF
ELECTROSPUN
POLYCAPROLACTONE (PCL)-SPIRULINA NANOFIBER AS A
SCAFFOLD FOR CELL CULTURE
Kim SH (1), Jung SM (1), Shin CH (1), Shin HS (1)
1. INHA UNIVERSITY
Scaffolds are important for pattern of cellular behaviors
in tissue engineering. In many scaffold fabrication
methods, Electrospinning is a simple technique to make
nanofiber mat that is similar to the natural extracellular
matrix structure. In recent year, it has been
demonstrated that electrospun nanofiber mat comprising
synthetic
biodegradable
polymer
such
as
polycaprolactone(PCL), poly l-lactide acid(PLLA) and poly
vinyl alcohol(PVA). Especially, PCL is a semi-crystalline
polyester that is a popularly used bio-polymer for tissue
engineering. However it has limited cell adhesion,
proliferation and differentiation because of their
hydrophobic property. . In this research we made PCL
nanofiber which contains a blue-green microalgae,
spilurina and examined some advanturous specialties for
cell culture and tissue engineering. We demonstrated
that spirulina-containing PCL nanofibrous scaffolds
enhances cell adhesion and proliferation in comparison
with PCL nanofiber
This work was supported by the Korea Science and
Engineering Foundation (KOSEF) grant funded by the
Korea government (MEST). (MEST 2010-0015308)
Keywords. electropinning spirulina polycaprolactone
(5.P3) DIRECTING BIOLOGICAL RESPONSE THROUGH
MATERIAL PROPERTIES
Kuforiji FO (1), Clemments M (2), Jenkins G (3), El Haj AJ
(1), Harts S (4), Roach P (1)
1. Institute for science and Technology in Medicine, Keele
University, ST4 7QB, UK; 2. School of life science,
University of Westminster, W1B 2UW, UK; 3.Centre for
Bio-Inspired Technology, Imperial College London, SW7
2AZ, UK; 4. School of life science, Keele university, ST5 5BG
Introduction. Biomaterials are used in tissue engineering
to repair, replace or augment healthy tissue. Although
mechanical properties derived from the material bulk are
well established, attention has turned towards the
surface of biomaterials in order to more easily integrate
these materials into the body. Cells naturally secrete
proteins in order to moderate their environment,
providing a route for many cellular mechanisms including
attachment and proliferation. To date, little information
on cellular mechanisms in relation to their interaction
with biomaterial surfaces has been reported. By
understanding such responses will allow for the
development of advanced biomaterial coatings,
controlling cellular responses with medical devices.
Materials and Methods. Mass spectrometry has been
used to evaluate differences in cellularsecretions in
relation to a range of surface chemistries. 3T3 fibroblasts
were cultured over surfaces presenting OH, COOH, NH2
and CH3 terminal chemistry, prepared using silane self
assembled monolayers on glass. Cell culture media was
taken at varying time points after cell seeding, being
worked up via acetonitrile precipitation and ZipTip
desalting procedures and analysed using electrospray
mass spectrometry.
Results. Mass spectral differences are found highlighting
variation in cell secretions in relation to their interaction
with the underlying surface chemistry. Cellular
morphology, adhesion and proliferation rates also show
varying responses of cells to surface chemistry.
Conclusion. 3T3 fibroblasts have been shown to adhere,
proliferate and have distinct morphology depending upon
the surface chemistry on which they reside. Differences in
secreted proteins were also observed indicating that
surface chemistry controls internal cellular processes.
We acknowledge funding from EPSRC DTC programme
and the National Endowment for Science, Technology and
the Arts (NESTA).
Keywords. Biomaterial, Protein, surface chemistry,
cellular reponse, Fibroblast
(5.P4) TISSUE-ENGINEERED HYPERTROPHIC CARTILAGE
UNDERGOES ANGIOGENESIS AND OSTEOGENESIS IN
CRANIAL DEFECTS
Kwarciak A (1), Bardsley K (1), Freeman C (1), Brook I (1),
Hatton P (1), Crawford A (1)
1. University of Sheffield
Introduction.Tissue-engineered hypertrophic cartilage
grafts have significant potential for the repair and
reconstruction of large bone defects. Hypertrophic
cartilage has the advantage over bone grafts in that it can
withstand the low oxygen levels typically found at the site
of injury, and it may also induce angiogenesis and
osteogenesis. Previous research showed that nasal
chondrocytes could form a hypertrophic-like cartilage,
but also that this tissue failed to mineralise in vitro.
Objective: To investigate the ability of tissue-engineered
hypertrophic-like cartilage to undergo mineralisation to
form bone tissue in vivo in a cranial defect in the rat.
Materials and Methods. Rat nasal chondrocytes were
cultured for 42 days on poly-glycolic acid (PGA) in
standard chondrogenic conditions. 3.5mm circles were
cut from the constructs and implanted into cranial
defects (3.5mm) of 12 week old Wister rats (n=8) for 4 or
8 weeks. On retrieval, calvaria were fixed in formalin for
analysis by µCT and paraffin embedded for histological
analysis. All animal experiments were carried out with the
relevant regulatory (Home Office) approval.
Results. By week 4, good infiltration of blood vessels was
seen throughout the construct and after 8 weeks
deposition of bone tissue was observed histologically.
Analysis by µCT showed small islands of mineralisation in
the constructs after 4 weeks. By 8 weeks bone formation
was significant with most of the defect filled with new
bone. Relatively little bone formation was seen in empty
defects by 8 weeks.
Conclusion. Tissue-engineered hypertrophic-like cartilage
grafts underwent angiogenesis and osteogenesis in vivo
and promoted healing of calvarial defects.
The work was performed as a part of the EXPERTISSUES
Network of Excellence (EC contract: NMP3-CT-2004500283) and funding was received from the Marie Curie
programme (Alea Jacta Est, EC contract MEST-CT-2004008104).
Keywords. Hypertrophic cartilage; endochondral
ossification; tissue-engineering
(5.P5) BIODEGRADATION BEHAVIORS OF SILK FIBROIN
MEMBRANE FOR REPAIRING OF TYMPANIC MEMBRANE
PERFORATIONS
Park CH (1), Lee OJ (1), Lee JM (1)
1. Hallym University
Silk fibroin of silkworms has been widely studied as
biomaterials. The degradation behavior of silk
biomaterials is obviously important for medical
applications. But the study about long- term result is few
in vivo. In this work, we investigated the degradation
behavior of silk fibroin membrane in vitro and in vivo. In
vitro assay, we observed degradation of silk membrane in
PBS, culture media and enzyme (protease K) solution. In
solution with protease K, 80% of silk membranes were
degraded within 10 days. Silk membranes presented no
cytotoxicity in L929 cells and rat tissue. In order to
investigate degradation of silk membrane in vivo, silk
membrane implanted subcutaneous in rats and were
harvested after surgery until 19 months. SEM, histological
analysis of silk membrane explants showed that silk
membrane broken in several pieces from 16 months. In
conclusion, the results indicated that silk membrane is a
good biocompatible and has a long degradation time as
biomaterials.
Keywords. silk fibroin, membrane, biodegradation
(5.P6) MICROPARTICLES AGGLOMERATED IN FIBRIN
GELS FOR CARTILAGE REGENERATION
Gamboa-Martínez TC (1), García-Cruz DM (2), CardaBatalla C (3), Gómez-Ribelles JL (1), Gallego-Ferrer G (1)
1. Centre for Biomaterials and Tissue Engineering,
Universitat Politècnica de València, Spain; 2. Regenerative
Medicine Unit. Centro Investigación Príncipe Felipe (CIPF),
Spain; 3. Pathology Department, Faculty of Medicine and
Odontology, Universidad de Valencia, Spain
Introduction. Recently the production of composite
injectable vehicles is a powerful alternative to avoid the
drawbacks of implantation of other forms of 3D scaffolds.
In this study, we developed a biodegradable composite
gel consisting of chitosan microspheres embedded in
fibrin gels as potential scaffold for articular cartilage
regeneration. The combination of both natural polymers
as cellular carrier promotes cell adhesion and allows a 3D
arrangement maintaining the chondrocytes in a
differentiated phenotype.
Materials and Methods. Chitosan microspheres (Cht
MCP) were made by a precipitation process. Chitosan
solution was dropped into a gelation solution (sodium
hydroxide 0.1M and absolute ethanol at a 70/30 volume
ratio) under continuous stirring. Gelled microspheres
instantaneously formed, were allowed to cure in the
gelation solution for 24 h. Finally, in order to prepare the
composites 2 wt. % fibrinogen solution was mixed with
microspheres and 1U/ml of thrombin solution. Those gels
were allowed to coagulate overnight at 37ºC, the formed
composite gel was finally crosslinked with 5 mM genipin
solution. Fibrin gel (fbn) without microspheres was used
as control. In the biological in vitro experiments human
chondrocytes were cultured for up to 4 weeks. Cellular
viability was assessed by live/dead cells kit, DNA
quantification and MTS assay. Chondrocyte morphology
and phenotype were examined by SEM and
immunofluorescence staining respectively.
Results. Figure 1 shows the cellular morphology over the
fibrin/ Cht MCP. The fibrin fibers covered the nanoporous
microspheres forming a smooth surface in the composite
and cells adhered firmly to the coated microparticles (a).
Chondrocytes remained viable at 14 days of culture (b). A
higher number of cells was found in the control samples
compared with composite structures. The limited
proliferation suggests that cells can maintain better their
phenotype avoiding fast dedifferentiation, despite
collagen type I expression (c) and actin cytoskeleton
development (d) detected in the samples.
Conclusions. Chondrocytes cultured in fibrin gels and in
fibrin/chitosan microspheres composite gels are viable
but some cells in hybrid materials show characteristics of
dedifferentiated chondrocytes.
The authors acknowledge the financial support of the
Spanish Ministry through the DPI2010-20399-C04-03
project and the funding of the Instituto de Salud Carlos III
and the CIPF for the “Investigación Básica y Traslacional
en Medicina Regenerativa”.
Keywords. gel, polysaccharide microspheres, fibrin,
articular cartilage
(5.P7) ULTRASTRUCTURE CHARACTERIZATION FOR
BONDING EFFICACY OF RESIN TO DENTAL CEMENTUM
Aguilera FS (1), Osorio E (1), Toledano M (1), Osorio R (1)
1. University of Granada, Spain
Introduction. Margins of dental adhesive restorations are
frequently located at the cementum or cervical outer
dentin. The root cementum has high organic content and
predominantly consists of cross-linked collagen structure,
less hard and more permeable compared with enamel
and dentin. New adhesive systems have been developed
in an attempt to obtain a reliable bonding to all tooth
substrates. The aim of the study was to determine the
bonding efficacy of three adhesive systems to human
cementum, and to assess the promoted surface
roughness.
Materials and Methods. Extracted human canines were
used for the present study. Cervical cementum surfaces
were ground flat with wet 600-grit silicon carbide paper
and bonded. Three adhesive systems were employed: an
etch-and-rinse adhesive (Single Bond –SB-), a two-step
self-etching (Clearfil SE Bond –CSEB-), and a 1-step selfetching (One-Up Bond F –OUB-) adhesive. After applying
the adhesive, resin composite build-ups were constructed
and stored in a humid environment for 24 h at 37ºC.
Specimens were sectioned into 1 mm2 beams and tested
for microtensile bond strength (MTBS). Additional
surfaces were conditioned for Atomic Force Microscopy
(AFM) analysis; digital images of treated surfaces (5x5 and
15x15 µm) and average surface roughness of the scanned
areas were obtained. Data were analyzed by ANOVA and
SNK multiple comparisons tests (p<0.05).
Results. Means and standard deviations of MTBS (MPa)
and roughness (Ra –nm-) values are shown in the table.
Letters and numbers show differences in columns.
5x5 µm (R
No treatme
35% H3PO4 et
+ SB
32.77 (11.
15x15 µm
74.15 (6.2
MTBS (M
--
51.79 (15.4 232.96 (35
51.49 (10.
CSEB
36.02 (4.6
62.42 (10.
21.14 (12.
OUB
20.84 (7.2
26.1 (2.7
31.40 (16.
Conclusion. When phosphoric acid treatment was
applied, cementum surface roughness increased and a
strong demineralization with exposed collagen fibers was
observed. The etch-and-rinse adhesive promoted highest
bond strength on human cementum surfaces.
CICYT/FEDER MAT2008-02347/MAT, JA-P07-CTS2568 and
JA-P08-CTS-3944.
Keywords. dental cementum, roughness, bond strength,
adhesive systems
(5.P8) MICROCOMPUTED TOMOGRAPHY ANALYSIS OF
BONE REGENERATION AFTER MAXILLARY SINUS
AUGMENTATION: A CASE REPORT
Meleo D (1), Pecci R (1), Corbi S (2), Soda G (3), Bedini R
(1)
1. Department of Technology and Health, ISS, Rome; 2. S.
Camillo-Forlanini Hospital, Rome; 3.Sapienza University,
Rome
Introduction. In the last few years bone tissue
regeneration studies has led to a better knowledge of
chemical and structural features of biomaterials. Scaffolds
for bone tissue engineering should provide a threedimensional design and an osteoconductive surface to
promote the ingrowth of new bone after implantation
into bone defects. The possibility of investigation on their
morphometric characteristics allows to evaluate the
predictability
of
regenerative
process.
X-ray
microtomography (microCT) is a miniaturized form of
conventional tomography, able to analyze in a noninvasive and non-destructive way the internal structure of
small objects, performing three-dimensional images with
high spatial resolution (<5 micron pixel size). The aim of
this work is to illustrate the possible applications of
micro-CT in the analysis of human bone grafted with
different scaffolds in order to obtain morphometric
parameters and three-dimensional reconstruction by
using the SkyScan 1072 scanner.
Methods and Results. We present a case of a patient who
needed a bilateral maxillary sinus lift for dental implants
placement. One side was grafted with a bovine
hydroxyapatite (Endobon, Biomet 3i) while the
controlateral sinus received a synthetic beta three
calcium phosphate (Cerasorb, Curasan). A bone sample
for each side was taken at implant placement surgery
(after about six months post sinus augmentation) and was
analyzed by microCT. Histologycal examination was also
performed to illustrate advantages and disadvantages of
microtomography versus traditional microscopy (fig. 1).
Conclusion. Since there is a close relationship between
the properties of a bioscaffold and its microstructure, it is
necessary to examine it using the highest level of
resolution before being able to improve existing materials
or to design new products. For a correct analysis, the
samples should not have been modified or treated in any
way, so the microCT is a non-invasive and non-destructive
technique and its appliance gives considerable results in
biomaterials’ studies and tissue engineering.
Keywords. scaffold, osteoconduction, microcomputed
tomography
(5.P9) FUNCTIONALIZATION OF 3D POROUS BONE
SUBSTITUTES USING LAYER-BY-LAYER TECHNOLOGY
Jacobi A (1), Schuba S (1), Arnold LY (2), Volodkin D (3),
Stiehler M (1)
1. University Hospital Carl Gustav Carus at Technical
University Dresden, Centre for Translational Bone, Joint
and Soft Tissue Research, Germany; 2. National University
of Singapore, Department of Bioengineering, Singapore;
3. Technical University Berlin, Department of Chemistry,
Applied Physical Chemistry, Germany
Introduction. To avoid donor site morbidity material
associated with autologous bone grafting and due to
limited availability of bone autograft, the use of
cancellous bone allografts (CBA) is an appealing strategy
for the therapy of localized bone defects.
Functionalization with osteogenically active factors may
enhance long-term osseointegration and improve the
clinical performance of CBA. The Layer-by-Layer (LbL)
method involves the formation of polyelectrolyte
multilayer (PEM) films whereby layers of oppositelycharged electrolytes are deposited alternatively. The aim
of this study was to establish a protocol for optimized
surface coating of 3D porous bone substitute materials
applying alternating deposition of hyaluronic acid (HA)
and poly-L-lysine (PLL) by the LbL method. The effects of
the number of deposition steps on the morphology,
proliferation and osteogenic differentiation of
mesenchymal stromal cells (MSC) were investigated.
Materials and Methods. Human MSC were cultured in
monolayer (2D) and on CBA scaffolds (3D) for up to 14
days. CBA scaffolds and cell culture plates were modified
by coating with PLL/HA films (n=12 and n=24).
Proliferation was assessed by total DNA quantification.
Osteogenic differentiation was evaluated by alkaline
phosphatase (ALP) activity. Coating density and cellular
distribution were performed by fluorescence microscopy
(FM).
Results. Proliferation rate of MSCs with and without the
use of PLL-HA films (n=12 or n=24) showed no significant
differences after 14 days. By 3D cultivation a decreased
proliferation was observed. 2D cultivation of MSCs
stimulated osteogenic differentiation as observed by
increased cell-specific ALP activity compared with 3D
cultivation on day 14. FM demonstrated that polymer
films were distributed homogenously throughout the CBA
samples and were stable for up to 14 days.
Conclusion. PLL-HA coating with n=24 is suitable for 2D
and 3D static cultivation of MSCs on CBA scaffolds.
Further studies will address the evaluation of LbLmediated growth factor functionalized CBA on
proliferation and osteogenic stem cell differentiation.
Keywords. bone substitutes, Layer-by-Layer technology,
MSCs
(5.P10) ZINC STABILIZES DENTIN COLLAGEN AFTER
ETCHING PROCEDURES.
Osorio E (1), Osorio R (1), Yamauti M (1), Quintana M (1),
Ruiz-Requena ME (1), Toledano M (1)
1. University of Granada
Introduction. Partial demineralization of a dentin
mineralized collagen by acids may represent a suitable
collagen to be remineralized, in the presence of minerals.
Demineralized exposed collagen can undergo degradation
by endogenous matrix metalloproteinases. Effective
inhibitors of matrix metalloproteinases may be included
in resin-dentin interfaces to protect the seed crystallitesparse collagen fibrils, from degradation before they
could be re-mineralized.
Materials and Methods. A human dentin beam
degradation assay was performed. Dentin beams were
obtained and included in: 1) demineralized beams
created by 10% phosphoric acid (PA); 2) demineralized
beams created by 0.5 M EDTA; 3) immersion of
mineralized beams in Clearfil SE Bond primer (SE); and 4)
immersion of mineralized beams in Xeno V (XE). Two
demineralized dentin (approx. 2 mg) were placed in each
incubation media: 1) artificial saliva -AS-; 2) 40 mM
chlorhexidine digluconate in AS; 3) 3.33 mg/ml of zinc
chloride in AS; 4) doxycycline (1:1) was added to the AS.
Dentin beam specimens were incubated in 500 µl of
media at 37oC for 24 h or 3 wk. Supernatants were
analyzed for the release of collagen degradation product
(C-terminal telopeptide of type I collagen -ICTP-) using a
radioimmunoassay. Values were analyzed by ANOVA and
SNK multiple comparison (P<0.05).
Results. Mean ICTP values and multiple comparisons
results are in the table. Identical numbers in each row
indicate no significant difference. In each column values
with identical low case letters indicate no difference
between solutions within the same dentin treatment and
identical upper case letters indicate no significant
difference between treatments within the same solution.
Conclusions. Zinc at high concentrations serves as potent,
stable and effective inhibitor of dentin MMPs. MMPs
degradation of collagen is strongly reduced in resin
infiltrated dentin and zinc addition lowered degradation
to values near to those of mineralized dentin.
CICYT/FEDER MAT2008-02347, JA-P07-CTS2568 and JAP08-CTS-3944.
Keywords. dentin, metalloproteinases, Zinc, inhibitor
Dentin treatment
PA-treated + AS
PA-treated + AS + chlorhexidine
PA-treated + AS + Zn
PA-treated + AS + Doxycycline
24 h
70.01 (16.67) 1a
30.82 (8.29) 1b
16.32 (5.84) 1c
<0.01 (0.00) 1d
C
C
C
A
3 weeks
178.23 (22.51) 2ab C
200.56 (16.55) 2a C
45.66 (5.98) 2c B
<0.01 (0.00) 1d A
160.34(16.32) 2a
179.88 (14.57) 2b
79.25 (8.06) 2c
<0.01 (0.00) 1d
EDTA-treated + AS
EDTA-treated + AS + chlorhexidine
EDTA-treated + AS + Zn
EDTA-treated + AS + Doxycycline
80.46 (8.70)
16.76 (1.40)
10.99 (1.61)
<0.01 (0.00)
1a
1b
1b
1c
C
B
B
A
C
B
C
A
SE-treated + AS
SE-treated + AS + chlorhexidine
SE-treated + AS + Zn
SE-treated + AS + Doxycycline
12.24 (1.11)
6.24 (0.33)
1.51 (0.11)
<0.01 (0.00)
1a
1b
1c
1d
A
A
A
A
49.97 (4.16)
30.38 (2.55)
5.28 (0.49)
<0.01 (0.00)
2a
2b
2c
1d
A
A
A
A
XE-treated + AS
XE-treated + AS + chlorhexidine
XE-treated + AS + Zn
XE-treated + AS + Doxycycline
27.96 (0.91)
6.64 (0.40)
2.39 (0.28)
<0.01 (0.00)
1a
1b
1d
1e
B
A
A
A
69.32(6.55)
32.33(2.14)
6.33 (0.56)
<0.01 (0.00)
2a
2b
2d
1e
B
A
A
A
(5.P11) EVALUATION OF CALCIUM PHOSPHATE
CERAMICS FABRICATED FROM EXTRACTED HUMAN
TEETH FOR TOOTH TISSUE ENGINEERING
Lim KT (1), Kim J (1), Seonwoo H (1), Chung JH (1)
1. Seoul National University
Bioceramic tooth powders were prepared via heat
treatment of extracted human teeth using sintering
temperatures between 600ºC and 1200ºC, and their
properties were investigated for potential tooth tissue
engineering. The sintered human tooth powders were
characterized using thermal analysis (thermogravimetric
analysis (TG) and differential thermal analysis (DTA)), field
emission scanning electron microscopy (FE-SEM), X-ray
diffraction (XRD), energy dispersive X-ray spectroscopy
(EDX), and Fourier transformed infrared (FTIR)
spectroscopy. Additionally, the phase constitutions and
chemical homogeneities of the composite samples were
examined using a quantitative chemical analysis with
inductively coupled plasma (ICP) spectroscopy. The
results revealed that the annealing process produced
useful hydroxyapatite-based bioceramic biomaterials
when annealed above 1000℃. The FTIR spectra and the
TG/DTA thermograms of the tooth powders indicated the
presence of organic compounds, which were completely
removed after annealing at temperatures above 1000℃.
The tooth powders annealed between 1000ºC and
1200ºC had good characteristics as bioceramic
biomaterials. Furthermore, the biocompatibility of each
tooth powder was evaluated using in vitro and in vivo
techniques; our results indicate that the prepared human
tooth powders have great potential for tooth tissue
engineering applications.
Keywords. human tooth powder, calcium phosphate
bioceramics, extracted teeth, regenerative medicine,
tooth tissue engineering
(5.P12) BONE HEALING USING TISSUE-ENGINEERED
CONSTRUCTS FOR LONG BONE CRITICAL SIZED DEFECTS
IN SHEEP
Viateau V (1), Manassero M (1), Sudre L (2), Oudina K (2),
Valentin M (2), Logeart D (2), Petite H (2), Bensidhoum M
(2)
1. Ecole Nationale Vétérinaire d’Alfort, 7 Av G. de Gaulle,
94700 Maisons Alfort, France; Laboratoire de
Biomécanique et Biomatériaux Ostéoarticulaire ; 2.
Laboratoire de Biomécanique et Biomatériaux
Ostéoarticulaire UMR 7052, 10 Av de Verdun, 75010 Paris,
France
Introduction. In a previous study, our group has shown
that standardized biohybrides engineered from Porites
(coralline scaffolds) and combined with autologous MSCs,
are efficient for bone critical-sized defect repair in sheep.
Several authors have emphasized that coupling scaffold
resorption and new bone apposition must be obtained for
bone healing. Using an ectopic sheep model, we have
previously compared the resorption and the bone
formation between Porites/MSCs and Acropora/MSCs.
This study has demonstrated a less extensive coral
resorption as well as a good bone formation in
Acropora/MSCs group. The aim of the current study was
to validate this promising biohybrid construct in a
clinically relevant bone defect.
Materials and Methods. Osteoperiosteal segmental
(25mm) defect was created in the left metatarsal bone
and the bone marrow was aspirated from the iliac crest of
each sheep. Each defect was filled with a poly-methylmethacrylate (PMMA) spacer. The PMMA spacer was
removed 6 weeks later and the defect was filled either
with Acropora scaffold (Group1, n=4) or with Acropora
scaffold loaded with autologous MSCs isolated from bone
marrow (Group2, n=5).
Results. 6 months after implantation, animals were
sacrified and each defect was assessed by microcomputed tomography and histology. No bone union was
observed in Group1, in contrast to Group2 where bone
continuity was observed in 4 sheeps. Acropora resorption
rate was higher in Group1 than in Group2 (99.2±0.8% v.s.
95.8±4.7%). The amounts of newly formed bone in
defects filled with coral/MSCs were significantly higher
from those filled with Acropora alone (677±227 v.s.
1357±471 mm3).
Conclusion. The present study is the first study evaluating
Acropora scaffold in an orthotopic model. This study
established clearly the benefits of using Acropora scaffold
loaded with MSCs for long bone defect in sheep.
Keywords. sheep, coral, Acropora, critical-sized-defect
(5.P13) EVALUATION OF A CORAL BONE SUBTITUTE IN A
NEW ORTHOTOPIC LARGE DEFECT IN MICE
Manassero M (1), Viateau V (1), Retortillo J (1), Matthys R
(2), Bensidhoum M (3), Petite H (3)
1.Ecole Nationale Vétérinaire d’Alfort, 7 Av G. de Gaulle,
94700 Maisons Alfort, France; Laboratoire de
Biomécanique et Biomatériaux Ostéoarticulaire; 2. AO
Research Institut, Innovations, Clavadelerstrasse 8, 7270
Davos, Switzerland; 3. Laboratoire de Biomécanique et
Biomatériaux Ostéoarticulaire UMR 7052, 10 Av de
Verdun, 75010 Paris, France
Introduction. Tissue-engineered bone constructs are an
appealing strategy to overcome drawback of autograft for
the treatment of massive bone defects. In mice,
preliminary evaluations of these types of constructs have
shown many advantages: low cost, homogeneity of
response, functional evaluation of biological processes.
However, large diaphyseal bone defect models in mice
are sparse and often use bone fixation which does not
provide optimal stability and which fills the bone marrow
cavity. The aims of this study were to develop a criticalsize segmental femoral defect in mice and to evaluate a
natural bone substitute in such model.
Materials and Methods. NUDE mice were used for this
study. A locking plate with 4 screws was applied on the
anterior femoral side. A 3-mm mid-diaphyseal ostectomy
was subsequently performed using a specific jig. The
resulting bone defects tested were as follows: Group A:
left empty (n=5); Group B: filled with a massive isograft
(n=6); and Group C: filled with massive coral scaffold
(Acropora) (n=6). Bone healing was assessed by
radiographs and micro-computed-tomography after
sacrifice, 9 weeks postoperatively.
Results. Stable bone fixation was maintained throughout
the study in all animals.
Bone union did not occur in Group A but were observed
in all animals belonging to Group B and C. Bone volumes
for Group A and B and volume of new bone formation for
Group C inner the defect were 0.78±0.3 mm3; 4.43±0.87
mm3 and 3.65±0.84 mm3, respectively. Results of Groups
B and C were similar, and significantly higher than those
of Group A.
Conclusion. The present study establishes a novel,
reproducible, murine large femoral defect (. Bone
bridging occurred in this model when a bone substitute
was used. This model allows further studies of the
molecular and cellular events that are involved in bone
replacement strategies especially with human cells.
Keywords. mice, coral, acropora, critical-sized-defect
(5.P14) ASSESSMENT OF MECHANICAL BEHAVIOUR OF
POTENTIAL IMPLANTS MADE OF POLYSILOXANE
CONTAINING LAYERED SILICATE NANOPARTICLES
Papadopoulos T (1), Tarantili PA (2), Vasilakos SP (2)
1. Biomaterials Lab., Dental School, University of Athens;
2. Polymer Technology Lab., School of Chemical Eng.
Introduction. Silicone rubber implants have long been
used for local contour corrections such as nasal, chin, and
cheek augmentation, because they are water-repellent,
heat stable and chemically inert materials. They are also
used in the manufacture of medical devices including
urological, orthopaedic, ophthalmic and drug delivery
devices, such as vaginal rings and transdermal implants.
However, most applications require that poly(dimethyl
siloxane) (PDMS) be reinforced by solid fillers because the
very poor mechanical strength of the unfilled elastomer.
Layered-silicate clays are interesting reinforcing agents
due to their high aspect ratio and exceptionally stable
oxide networks.
Materials and Methods. Condensation type PDMS, grade
DMS-S31 (Gelest Inc.), was the elastomer matrix and
organic modified montmorillonite (OMMT) under the
trade name Cloisite® 20A (Rockwood Clay Additives
GmbH)
the
reinforcement.
PDMS/organoclay
nanocomposites were prepared using the sonication
technique and were characterized by X-ray diffraction
(XRD), hardness measurements (Shore A), tensile and tear
tests. The solvent uptake of immersed PDMS
nanocomposites was also measured at 25oC.
Results. The XRD analysis showed that within the
experimental setup of this work, for OMMT
concentrations up to 5 phr, intercalated/exfoliation
hybrids
are
formed.
The
incorporation
of
montomorillonite significantly improves the tensile
properties of PDMS matrix. Increase in tearing strength
and hardness of PDMS nanocomposites was also
observed in comparison with the unfilled elastomer. The
increase of clay content decreases swelling of the
elastomer due to an increase in the tortuosity, because of
the presence of clay particles, whereas an increase in the
crosslinking density can be observed, probably due to
physicochemical interactions between the organoclay
reinforcement and polysiloxane molecules.
Conclusion. The incorporation of OMMT in silicone
rubber significantly improves its performance properties
assessed in terms of hardness, as well as tensile and tear
strength. This suggests that the studied hybrids would
display improved behaviour when used as implants in
biomedical applications.
Keywords. polysiloxane elastomers, layered silicate,
nanocomposites, implants
(5.P15) TRIPPLE CONFIRMATION FOR BIOACTIVITY OF
SYNTHETIC HYDROXYAPATITE (HAp) IN BONY
ENVIRONEMENT
Skagers A (1), Salma I (1), Pilmane M (1), Salms G (1),
Feldmane L (1), Neimane L (1), Berzina-Cimdina L (1)
1. Riga Stradina University
Introduction. To evaluate reactogenicity of contacting
soft and bone tissue after implantation of synthetic HAp
bioceramics in experimental and clinical conditions.
Material and methods. HAp ceramic implants with
porosity of ceramics 25% – 30 %, on 14 rabbits were
inserted in bone, on 12 animals subperiostaly . After two
weeks and three months expression of TGFß and
apoptotic cells were evaluated. Late outcomes of
maxillary sinus floor elevation with HAp granules and 278
SEMADOS (BEGO) dental implant insertion on 185
patients were evaluated using also cone beam 3D
method. In 31 cases biopsy of biomaterial/host tissue
hybrid and alveolar bone was done.
Results. Two weeks after intraossal implantation
expression of TGFß in bone around HAp implants was in
marked number of bone cells while in control side was no
expression. After subperiostal implantation in two weeks
was no expression of TGFβ either in bone or soft tissue,
after three months was rich expression in both bone and
periosteum. Apoptotic cells were in moderate number
around HAp implants. Radiodensitometry of the elevated
sinus floor area showed 98 % after operation, 82%
average after 6 months, 76 % after 3 years of loading and
65 % after 5 years. Radiodensity of residual alveolar bone
was 55 % after the implantation, 64 % at second stage
surgery, 78% after 3 years and 70 % after 5 years. In
biopsies after 6 months were bone trabecules, fibrous
tissue and granules with degradation by osteoclast – like
cells without inflammatory cells.
Conclusion. Release of endogenous growth factor,
remodeling of HAp / host hybrid and contacting atrophic
bone remineralisation confirm bioactivity of synthetic
porous HAp biomaterials in bone environment.
Key words. HAp bioceramics, transforming growth factor
β, biopsy, radiodensitometry.
Supported by Latvia State programm’s Project „New
materials and technologies for substitution of biologic
tissue”.
(5.P16) RESPIRATORY GATED MICRO COMPUTED
TOMOGRAPHY FOR IMPLANTATION SITE IMAGING
DURING IN-SITU TISSUE ENGINEERING IN LIVE SMALL
ANIMAL MODEL
Saxena AK (1), Soltysiak P (1), Höllwarth ME (1)
1. Medical University of Graz
In our attempts to tissue engineer the esophagus using
the in-situ tissue engineering approach, investigations
were performed using the Sprague-Dawley rat model
using respiratory gated micro CT to observe implanted
constructs. Rat esophageal epithelial cells (REEC) were
isolated and cultured. The cells were seeded 14 days after
isolation on collagen scaffold disks. The cell-scaffold
construct were fabricated into tubes using a silicon stent
using Polyglytone-6211 monofilament absorbable
interrupted sutures and implanted into the rat omentum.
Under general anesthesia, a laparotomy was performed
and the omentum exposed. During 6 months of in-situ
tissue engineering, repetitive imaging of the abdomen
was performed using the Inveon® MicroCT equipment
(Siemens Medical, Malvern, PA, USA) with the Isoflurane®
system and a 125mm detector with a voxel size of
35.55µm. A further advantage of using this device is the
respiratory and cardiac gating capability of the rodent
being investigated. This is possible through a high-speed
shutter that acquires image frames with exposure times
as short as 10ms for cardiac and respiratory gated studies
with 4 TTL gating ports allowing management of dynamic
acquisition. For imaging, the rat was first put into the
anesthesia induction chamber, which was connected to
the Isoflurane® small animal vaporizer that delivers
accurate concentrations under varying conditions of flow
rate and temperature, particularly at low flows. For
induction the concentration of the anesthetic gas was set
on 5%, after which the rat was transferred into the
Inveon® MicroCT system. Tubes from the vaporizer were
then connected to the anesthesia inputs in the MicroCT
device with dedicated rat naso-mouth anesthetic mask
placed on the animal.
MicroCT of constructs undergoing in-situ tissue
engineering is a non-invasive method for imaging of
implanted constructs when a live small animal model.
(5.P17) PCK-26 ANTIGEN EXPRESSION IN ADULT AND
FETAL OVINE ESOPHAGEAL EPITHELIAL CELLS IS NOT AN
INDICATOR
OF
END
CELL
DIFFERENTIATION:
IMPLICATIONS FOR ESOPHAGEAL TISSUE ENGINEERING
Saxena AK (1), Ainoedhofer H (1), Kofler K (1), Höllwarth
ME (1)
Introduction. In esophageal epithelium, cells move from
the basal layer towards the lumen demonstrating
increased differentiation and reduced proliferation
capabilities. The aim of this study was to investigate the
expression of cytokeratin in adult ovine esophageal
epithelium (AOEE) and fetal ovine esophageal epithelium
(FOEE) and to determine if cytokeratin expression also
represented cells with proliferative potential.
Materials and Methods. Biopsies of ovine esophageal
epithelium (OEE) were obtained from fetal lambs in late
pregnancy (120 days) and were compared to those from
adult sheep. FOEE and AOEE were investigated using pancytokeratin antigen (PCK-26) and Proliferating Cell
Nuclear Antigen (PCNA) markers. Furthermore, PCK-26
positive esophageal epithelial cells (EEC) were isolated
using Fluorescent Activated Cell Sorting (FACS) and
analyzed for PCNA expression to estimate their
percentage in AOEE.
Results. PCK-26 expression was prominent in AOEE but
reduced in FOEE. PCNA expression was found throughout
the FOEE, however was limited toward the AOEE basal
layer. PCK-26 positive EEC with PCNA expression
accounted for 24% of the total cells in AOEE.
Conclusion. PCK-26 antigen is not a marker of
differentiation as it also represents cells with high
proliferative capability in AOEE. FOEE in late gestation
also demonstrate weak PCK-26 antigen expression but a
high expression of proliferation.
Keywords. cytokeratin, expression, proliferation
(5.P18) ESOPHAGEAL SMOOTH MUSCLE CELLS
DEDIFFERENTIATE WITH LOSS OF Α-ACTIN EXPRESSION
AFTER 8 WEEKS IN-VITRO CULTURE: IMPLICATIONS ON
ESOPHAGUS TISSUE ENGINEERING
Saxena AK (1), Ainoedhofer H (1), Tausendschon J (1),
Kofler K (1)
Introduction. Esophagus tissue engineering using the
hybrid-construct approach which involves assembly of the
various esophageal components and amalgamating them
to engineer the organ are presently being investigated.
The aim of this study was to investigate the
dedifferentiation and loss in expression of esophageal
smooth muscle after explant expansion in culture.
Materials and Methods. Ovine esophagus smooth muscle
cells (OESMC) were sourced from adult biopsies and
expanded in culture using the explant technique. The
explants were maintained under static in-vitro tissue
culture conditions with medium changes performed on
every 2nd day. Flow cytometry analysis was performed
for α-smooth muscle actin (α-SMA) expression at
intervals of 4 weeks for up to 8 weeks.
Results. OESMC reached confluence after 3 weeks in
culture and sufficient cells could be obtained after 4
weeks expansion to permit flow cytometry. After 4 weeks
58.5% OESMC exhibited α-SMA which decreased to 28.5%
after 6 weeks in culture. After 8 weeks in culture a mere
1.3% OESMC demonstrated α-SMA expression.
Conclusion. OESMC proliferated using the explant
technique exhibit a total loss of α-SMA expression after 8
weeks of in-vitro culture. The data obtained from these
investigations are crucial for tissue engineering of the
esophagus using the hybrid-construct approach.
Keywords. smooth muscle, expression, culture
(5.P19) BIOARTIFICAL TISSUE FOR THE SURGICAL
RECONSTRUCTION OF TRACHEAL LESIONS
Dally I (1), Pusch J (2), Hansmann J (2), Schandar M (2),
Linke K (2), Walles T (3), Walles H (3,4)
1. Institute for Interfacial Engineering, University of
Stuttgart IGVT; 2. Fraunhofer Institute for Interfacial
Engineering and Biotechnology, IGB; 3. Schillerhoehe
Hospital & Fraunhofer Institute for Interfacial Engineering
and Biotechnology, IGB; 4. Tissue Engineering and
Regenerative Medicine, University Würzburg
Introduction. Many graft materials and transplantation
approaches have been developed to create a clinically
applicable tracheal substitute. However, all these
substitutes are lacking a vascular system resulting in
insufficient oxygen and nutrient supply and ultimately
graft necrosis. Here, we present GMP-conform
techniques to generate a human, non-immunogenic,
tissue equivalent with an innate vascularization that can
be used as bioartifical tracheal graft.
Materials and Methods. We improved a well-established
protocol to generate a decellularized and DNA-free
vascularised scaffold from porcine jejunal segments,
which exhibits an arterial and venous pedicle as well as
the former luminal structures. Decellularization state,
DNA content and endotoxine levels were analyzed by
routine histological staining (H&E) and the use assays
against gallic acids and a limulus amebocyte lysate
assay.Human microvascular endothelial cells were seeded
into the capillary system whereas primary fibroblasts and
muscle cells were placed into the luminal structure.
Culture of the scaffold was performed in a custom-made
bioreactor module under dynamic culture conditions
corresponding to the human circulation. After 14 days of
culture,
we
performed
H&E
as
well
as,
immunohistochemical staining (Anti-fibroblast, CD31,
vWF, desmin and myoglobin). A vitality assay (MTT) was
employed to analyze the reseeding efficiency.
Results. Analyses of the scaffold revealed the complete
decellularization with endotoxin values consistent with
the prescriptive levels of the European Pharmacopeia.
After 14 days of culture within the dynamic bioreactor
system, confluent reseeding was observed by the use of
vitality assay and histological staining. Furthermore,
cellular identify was characterized using defined cellular
markers for each cell population.
Conclusion. In summary, we established an efficient
GMP-conform process for tracheal graft engineering
according to the requirements of the German Drug Act.
Currently we perform in vivo experiments to evaluate the
risk of vascular thrombosis. After we have received the
manufacturing licence, we aim to start clinical trials.
Keywords. bioartificial transplant, Trachea, GMP
Conclusion. Microscopy studies have demonstrated that
both, in gel and porous composites, collagen interacts
with arnica derived polysaccharides-coated liposomes
suggesting their use as intra-articular drug delivery
system.
Acknowledgements. This work was supported by Project
Cartbiotech, No. 62-059/2008.
Keywords. liposomes, Arnica montana derived
polysaccharides, collagen, articular drug delivery
(5.P20)
LIGHT
AND
ELECTRON
MICROSCOPY
CHARACTERIZATION OF A NEW COMPOSITE: COLLAGEN
AND ARNICA DERIVED POLYSACCHARIDE-COATED
LIPOSOMES
Zarnescu O (1), Moldovan L (2), Trif M (3), Moisei M (4),
Gaspar A (1), Craciunescu O (1)
1 .Faculty of Biology, University of Bucharest, Romania; 2.
National Institute of Research and Development for
Biological Sciences, Bucharest, Romania; 3 .Institute of
Biochemistry, Bucharest, Romania; 4. Institute of
Biochemistry, Bucharest, Romania
Introduction. Clinical studies have shown that intraarticular administration of anti-inflammatory drugs
encapsulated in liposomes shows prolonged residence in
the joint and reduction of inflammation. Moreover, the
anti-inflammatory properties of Arnica montana are very
useful for the treatment of osteoarthritis and rheumatoid
arthritis. The collagen-liposomes conjugates were found
to deliver higher levels of active agent over a sustained
period of time, in vivo, compared to normal collagenous
preparations.
The aim of this study was microscopy characterization of
a new composite based on collagen and Arnica montana
derived polysaccharides-coated liposomes, in order to use
it in the local treatment of rheumatic and inflammatory
disorders.
Materials and Methods. Liposomes were prepared using
the thin-film hydration method, followed by sonication.
Briefly,
a
mixture
of
phosphatidylcholine:
dioleoylphosphatidylethanolamine:
cholesterol:
stearylamine (4.125 mg lipids/ml; 4:2:3:1 molar ratio) was
dissolved in chloroform/methanol solution (95:5) and a
thin, dry film of these lipids was made in a rotary
evaporator. The film was hydrated with phosphate
buffered saline pH 7.4 containing Arnica montana derived
polysaccharides (4 mg/ml). In order to fabricate the
composite, a solution of collagen type I (5.3 mg/ml) was
mixed with a solution of polysaccharides-coated
liposomes, in a ratio of 1:1(v/v). The mixture was gelled at
room temperature. Gels were frozen at - 40 0C and
freeze-dried 24 h, for obtaining porous composites. Both,
gel and porous composites were used for light and
ultrastructural studies.
Results. In the presence of collagen type I,
polysaccharides-coated liposomes were entrapped within
the fibril network. Moreover, collagen fibrils appear to be
firmly attached to the liposome surface, suggesting the
existence of interaction between collagen and liposome
membrane.
(5.P21) FABRICATION AND CHARACTERIZATION OF
THREE DIMENSIONAL SCAFFOLDS OF BIOCERAMICPOLYMER COMPOSITE VIA MICROSTEREOLITHOGRAPHY
TECHNIQUE
Talib M (1), Covington JA (1), Smith (2), Grover L (2)
1. University of Warwick; 2. University of Birmingham
Microstereolithography is a method used for rapid
prototyping of polymeric and ceramic components. This
technique converts a computer-aided design (CAD) to a
three dimensional (3D) model, and enables layer per layer
fabrication curing a liquid resin with UV-light or laser
source. The aim of this project was to formulate a
photocurable polymer reinforced with calcium
pyrophosphate (CPP) and fabricate a scaffold for
application in tissue engineering. The photopolymer or
UV curable ceramic suspension was prepared with an
acrylate polyester, multifunctional acrylate monomer
with the addition of 50-70wt% of CPP, and
photoinitiators. From layer controlled determination, 3
wt% and 0 photoinniators was required to control an
effective area of localized photopolymerization, this also
depends on the weight fraction of CPP in the suspension.
The 3D structure of the photopolymer resin was
successfully fabricated using (µSL) apparatus (Envisiontec
Perfactory3® Desktop System). The resin were fabricated
in ‘dumb-bell’ form for tensile testing and a rectangular
prism shape specifically designed for 4 point bending, and
hardness measurement. They were then sintered at high
temperature for polymer removal, to obtain a ceramic of
the desired porosity. Morphology and CPP content of the
sintered polymer was investigated with SEM and XRD,
respectively. The addition of CPP coupled with high
temperature sintering, had a significant effect on the
mechanical properties exhibited by the bioceramic. The
density increased to more than 35% and the dimensional
shrinkage after sintering were 33%. The success
fabrication of novel bioceramic polymer composite with
µSL technique offer the possibility of designing complex
tissue scaffolds with optimum mechanical properties for
specific tissue engineering applications.
Keywords. Biomaterial, microstereolithography, calcium
phosphate, tissue engineering
6. BIOMATERIALS AND THE REACTIONS
THEY ELICIT IN THE BODY
Chair: Yvonne Bastiaansen-Jenniskens
Co-chair: Yves Bayon
Keynote speaker: Ruud Bank
Organizer: Yvonne Bastiaansen-Jenniskens
Synopsis: Biomaterials are very often used as scaffold for
regenerating tissue, either in vitro and to be implanted
later, or directly into the defect in vivo. A successful
biomaterial will integrate in the body without causing
massive inflammation and/or fibrosis. Inflammation is a
protective attempt by the organism to remove the
injurious stimuli as well as initiate the healing process for
the tissue. When this healing process is out of balance,
fibrosis can occur. Fibrosis can be designated as an
abnormal healing process characterised by an excessive
accumulation of extracellular matrix proteins (in
particular collagen). This process alters the extracellular
matrix structure and will eventually result in loss of
function of the particular tissue.
In this symposium we want to discuss the effects
biomaterials have on the behaviour of the cells seeded on
or surrounding the biomaterial focussing on reactions
related to inflammation, wound healing and fibrotic
reactions.
(6.KP) THE FOREIGN BODY REACTION AGAINST GELATIN
AND (NON-)CROSSLINKED COLLAGEN DISPLAY MAJOR
DIFFERENCES
Bank RA (1), Harmsen MC (1), van Putten SM (1)
1. Department of Pathology & Medical Biology, University
Medical Centre Groningen
Like any other biomaterial, implanted collagen scaffolds
induce a series of damage-inflicted processes that include
wound healing, inflammation and the foreign body
reaction (FBR). Macrophages play a pivotal role in the
tissue response. These macrophages interrogate the
biomaterial surface, release proteolytic enzymes and/or
phagocytose
the
biomaterial.
Under
certain
circumstances, the macrophages may fuse, to form
multinucleated foreign body giant cells. Collagen-based
biomaterials can be cross-linked to enhance the stiffness
and to dampen the rate of biological degradation. In
addition, non-crosslinked (native) collagen as well as
denatured collagen, i.e. gelatin, can be used. It is the
specific application that determines the choice of the
biomaterial. Biomaterial applications must take the tissue
response towards biomaterials into account.
Despite the frequent use of collagen-based scaffolds in
tissue engineering, remarkably little is known about the
nature of the foreign body reaction and the molecular
mechanisms that are involved in the breakdown of the
scaffolds. In a series of experiments, we observed that
the tissue response towards the gelatin disks and the
(non-)crosslinked, native collagen disks differs markedly
with respect to the number of macrophages, the
efficiency of giant cell formation, the size of the giant
cells, the influx of neutrophils, and the microenvironment (presence of IL-13 and TIMP-1), the
expression of MMPs and cathepsin K, and the expression
of the collagen receptors Endo180 and DDR-2. Thus, the
physical state of the collagen itself (denatured or native)
as well as its chemical nature (type of cross-link) has a
dramatic impact on the outcome of the foreign body
reaction. We will discuss the observed findings in terms of
degradation rates of the scaffolds and the mechanisms
involved in this degradation. In addition, we will show
that macrophages inside and outside the biomaterial
have different phenotypic properties.
Keywords. collagen, macrophages, foreign body reaction,
degradation
(6.O1) GENE EXPRESSION PATTERNS IN OSTEOGENIC
CELLS TREATED WITH STRONTIUM-SUBSTITUTED
BIOACTIVE GLASSES
Gentleman E (1), Autefage H (1), Park G (1), Stevens MM
(1)
1. Imperial College London, Department of Materials and
Institute of Biomedical Engineering
Introduction. Bioactive glasses (BG) are used as bone
replacements because they bond with living tissue and
dissolve upon implantation, releasing ions that stimulate
bone formation. Strontium (Sr) ranelate is an antiosteoporosis drug that works via Sr cations, which
stimulate osteoblast differentiation and prevent
osteoclast-mediated bone resorption. We have previously
shown that BG in which Ca was substituted with Sr,
promote osteoblast proliferation and activity and
decrease osteoclast activity and resorption. Here, we
examine the effects of Sr-substituted BG on gene
expression patterns in cultures of human mesenchymal
stem cells (hMSC) and primary osteoblasts (hOB).
Methods. BG in which 0, 10 or 100% of Ca was
substituted with Sr were produced. Culture media was
created by soaking with BG particles to release their
active ions. hMSC and hOB from 3 separate donors were
treated with BG-treated media for up to 14 days. RNA
was isolated and gene expression patterns were analysed
by quantitative real-time RT-PCR and whole genome
microarray.
Results and Discussion. We demonstrate that genes for
bone-specific transcription factors and proteins are
upregulated in cultures treated with BG compared to
controls treated with basal medium. In osteoporosis
patients treated with Sr ranelate, an anabolic effect on
bone formation has been observed. Here, we show that
osteogenic genes are upregulated to a greater extent in
hMSC and hOB treated with Sr-substituted BG compared
to standard all Ca BG controls. Taken together, these
results suggest that Sr-substituted BG upregulate key
genes in bone development, suggesting that it may be
possible to reproduce the anabolic effect on the skeleton
produced by orally delivered Sr ranelate in a biomaterial
that releases Sr locally. More extensive data analysis of
microarray results may also reveal insights into the
mechanism by which Sr acts on osteogenic cells.
Keywords. Bioactive glass, strontium, bone regeneration.
(6.O2) THE ROLE OF HYDROLYTIC ENZYMES AND
REACTIVE OXYGEN SPECIES IN AN IN VITRO MODEL OF
MACROPHAGE-MEDIATED
DEGRADATION
OF
POLY(TRIMETHYLENE CARBONATE) NETWORK FILMS.
van Kooten TG (1), Bat E (2), Kuijer R (1), Grijpma DW (3)
1. UMCG Groningen, Department of Biomedical
Engineering, The Netherlands; 2. University of Twente,
Molecular NanoFabrication Group, The Netherlands;
3. University of Twente, Department of Biomaterials
Science and Technology, The Netherlands.
Resorbable polymers are used in the human body as drug
carriers, as scaffolds for tissue regeneration, and in
preparation of degradable implants such as sutures.
Macrophages play an important role in the degradation of
these polymers. Enzymes and reactive oxygen species
(ROS) were shown to be involved in the degradation
process. This research aims at elucidating the
involvement of enzymes and/or ROS in the degradation
mechanism of gamma irradiated poly(trimethylene
carbonate) (PTMC) films.
The roles of enzymes and ROS in degradation were
evaluated by culturing murine J774 macrophages on
PTMC network films containing inhibitors for specific
degradation pathways. The influence of complement on
the degradation process was assessed as well.
Degradation was quantified by determining mass loss of
the PTMC discs. Macrophage activity was measured
through cytokine release of Il-6, MCP-1 and MIP-1α as
determined with ELISA. Cell coverage was calculated from
images obtained with confocal microscopy.
Cholesterol esterase was found to be the main
contributor to degradation as assessed by the inhibition
of degradation by diethyl umbelliferyl phosphate. The
results furthermore demonstrated that acid proteases
(inhibited by pepstatin A), serine and cysteine proteases
(inhibited by phenylmethyl sulfonyl fluoride) and ROS
(indirectly inhibited by apocynin through NADPH oxidase
and nitric oxide synthase) contribute less to the
degradation of PTMC networks. Activity of macrophages
was high both with and without the influence of
inhibitors, as indicated by high concentrations of secreted
cytokines MCP-1 and MIP-1α. Degradation in media
without complement was higher than in media with
complement.
The presented macrophage culture model is helpful in
reducing the number of animal experiments and provides
a useful fast, in vitro model to investigate the mechanism
of in vivo degradation of biodegradable polymers.
Keywords. macrophage, model, biodegradation, pTMC,
degradable
(6.O3) INNOVATIVE IN-VITRO POLYCULTURE MODEL, AS
AN ARTIFICIAL LIVING PERITONEUM, FOR ABDOMINAL
MESH EVALUATION
Lefranc O (1), Vernier A (1), Barrier F (1), David L (2),
Frank L (2), Siali R (2)
1. Covidien; 2. Therapol
Introduction. In-vitro assays are unavoidable in the
medical device evaluation process, and often represent
the first step for biomaterial characterization. However, it
is today well accepted that in-vitro cell culture assays are
non relevant of real life conditions.
Materials and Methods. In this study, a coculture model
involving the cells present in a healthy peritoneum was
developed. Human fibroblasts, macrophages, mesothelial
cells and/or endothelial cells were seeded in a type I
collagen matrix and cocultured up to reach a stable living
structure. The model showed baseline cytokine
expression which was dramatically increased when a
wound was induced on the surface.
Polypropylene (PP), Polyester (PET) and collagen coated
polyester (PETc) prostheses, presenting increasing
hydrophilicity gradients, were deposited on the coculture
model to evaluate the model different reactions when in
contact with those materials.
Results and discussion. To generate a wound, scalpel cuts
were applied on the coculture model inducing dramatic
pro-inflammatory cytokine secretion.
As already observed under single cell culture, the
coculture models still showed better cell adhesion and
proliferation on prostheses following hydrophilicity
gradients (PET and PETc) when compared with
hydrophobic prostheses (PP).
Furthermore, the tri-cells models presented a measurable
shrinkage (30%* in surface, *p<0,05) as reaction to the
bare prosthetic materials (PP and PET) while no model
shrinkage was measured at all for the collagen coated
prostheses (PETc), showing the collagen benefit for the
device integration. No shrinkage at all could be measured
when endothelial cells were added to the coculture,
highlighting physiological contractile reactions.
Conclusion. A new in-vitro complex coculture model was
developed as a living peritoneum. This model presented
better cell compatibility correlated with surface
hydrophilicity gradients and highlighted the collagen
positive impact on the in-vivo integration reaction.
Keywords. in-vitro, inflammation, mesh
(6.O4) HYDROPHILIC RESORBABLE AND BIOCOMPATIBLE
POLYMER SYSTEMS AS BIOACTIVE COATINGS OF
POLYPROPYLENE MESH AND CONTROLLED RELEASE OF
ANTIBIOTICS FOR TISSUE INTEGRATION
Fernández-Gutiérrez M (1), Olivares E (2), Bellón JM (2),
San Román J (1)
1. Institute of Polymers, CSIC. Juan de la Cierva 3, 28006 –
Madrid, Spain CIBER-BBN; 2. University of Alcala de
Henares, Spain CIBER-BBN
Introduction. The reparative process of hernia defects are
in general based on the apposition of polypropylene
meshes as biostable implants, which guarantees the
biomechanical stability of the affected tissue or organ.
After more than 30 years of clinical application, it is clear
that one critical point is the infection of the tissues or
organs in contact with the mesh and the consequences of
the infection process reaching statistical values around
20% in a period of 2 or 3 months after implantation,
depending on microorganism strain origin of the infection
process. In this work we present a novel an excellent
results on the application of bioactive and resorbable
hydrogel polymers based on copolymers of hydroxyethyl
methacrylate HEMA and 2-acrylamide- 2-methyl
propanesulfonic acid AMPS, as bioactive coating of
lightweight polypropylene PP meshes, and the addition to
the polymer system of a well known antibiotic,
vancomycin at a concentration of 20 wt-% respect to the
coating of the polymer applied.
Materials and Methods. The coating of the PP mesh was
obtained by the deeping of the mesh in a solution of 10 %
of copolymer with a composition 20 mole-% of AMPS and
80 mole-% of HEMA containing 20% of vancomycin. After
drying a homogeneous coating of 2.0 µm was obtained.
The antibacterial activity of the coated meshes was tested
by analyzing the inhibition areas of proliferation of agar
plates inoculated with Staphylococcus aureus SA or
S.epidermidis SE. The bioactivity was analyzed in vitro
using fibroblast cultures, and in vivo by implantation of
coated meshes in infected areas of the dorsal muscle of
rabbits, and analysis of the prosthesis and surrounding
tissue after 14 and 30 days of implantation.
Results. Results of the in vitro test demonstrated the
antibacterial activity of the coated meshes after 14 and
30 days, and an excellent correlation with the activity in
vivo after implantation during the same period.
The Figure shows the structure and morphology of the
mesh with the homogeneous coating of the bioactive
polymer containing 0.32 mg/cm2 of vancomycin. This
concentration is enough to control the infection without
detect vancomycin in the blood flow.
Conclusions
Quantitative evaluation of the release of vancomycin in
the animal model, as well the antibacterial activity of the
meshes in vitro and in vivo demonstrate that the
application of the coating offers excellent opportunities
to improve the behavior of PP meshes in clinical
applications with a minimum dose of antibiotic applied an
release in the point of infection and activity.
Keywords. synthetic polymers, drug delivery, bacteria
adhesion
(6.O5) DIFFERENTIATION OF MACROPHAGES INTO PROOR ANTI-INFLAMMATORY/ REPAIR SUBTYPE IN CULTURE
Grotenhuis N (1), Bayon Y (2), Falke L (1), Lange JF (1), van
Osch GJVM (1), Bastiaansen-Jenniskens YM (1)
1. Erasmus MC, University Hospital Rotterdam; 2.
Covidien-Sofradim Production, Trevoux, France.
Introduction. Macrophages are key immune cells in the
reaction to a biomaterial as they interact with proteins
adhered to biomaterials after implantation. To investigate
their specific response to biomaterials, it is important to
characterize macrophages in detail. Macrophages can be
roughly divided into a pro-inflammatory (M1) subtype
and an anti-inflammatory/repair (M2) subtype. The goal
of our study was to characterize macrophages
differentiated towards M1 or M2 phenotype and examine
the stability of this differentiation.
Materials and methods. Monocytes were isolated from
the blood of healthy donors using Ficoll separation and
magnetic cell sorting (MACS) based on magnetic
antibodies against CD14. Monocytes were stimulated for
one week with cytokines; LPS or IFN-γ for M1-stimulation,
dexamethasone or IL-4 for M2-stimulation. After
stimulation, cytokines were removed and culture was
continued for 7 days. Gene expression for IL-6, TNF-α and
IL-10 and morphology were analyzed at 0, 1, 3 and 7 days
after removal of the stimuli.
Results. Macrophages stimulated with LPS or IFN-γ (ie
M1- stimulated) had an elongated shape and expressed
high levels of IL-6 and TNF-α. Macrophages stimulated
with dexamethasone or IL-4 (M2-stimulated) had high IL10 gene expression and these cells had a round
morphology. After removal of the stimuli, differences
between M1- and M2-stimulated macrophages remain
for at least 7 days for all parameters.
Conclusion. Using soluble factors, macrophages can be
differentiated into a pro- or anti-inflammatory/repair
subtype, as characterized from selected read-out
parameters. Based on these parameters, this
differentiation appears to be stable for at least 7 days.
With this knowledge, differentiated macrophage
populations can be further used for the evaluation of the
inflammatory properties of implantable biomaterials, in in
vitro cell systems.
Keywords. macrophages inflammation biomaterials
(6.O6) TEXTURAL PROPERTIES AND IN VIVO RESPONSE
OF
CALCIUM
PHOSPHATES
CEMENTS-BLOOD
COMPOSITE
Mellier C (1), Fellah BH (2), Gauthier O (2), Rochet N (3),
Bujoli B (1), Janvier P (1), Bouler JM (2)
1. CEISAM CNRS UMR 6230, University of Nantes, France;
2. LIOAD INSERM UMR 791, University of Nantes, France;
3. CNRS 6235 GéPITOs, University of Nice, France.
Introduction. Calcium phosphates cements (CPCs) are
used as bone substitutes because of their similarity to the
mineral phase of bone. However, they can be considered
as fragile materials and they usually present limited
osteoconductive properties. So they still are dedicated to
fill small bone defects in non-load bearing conditions. The
purpose of this study was to investigate how blood
addition can interfere with setting processes and final
properties of two types of apatitic CPCs, one presenting a
shorter setting time (SST) and the other one a longer
setting time (LST).
Results and Discussion. α-TCP was transformed into
poorly crystalline apatite in all tested samples after 72
hours of incubation in a saline solution. For blood/LST
cement, both adhesion properties and time of workability
were significantly increased. Compressive strength tests
showed a ductile material behavior (fig 1.A). Regarding to
blood/SST cement (fig 1.B) only a slight increase of both
properties and time of workability was observed. After 12
weeks of implantation we could observe an excellent
bone/implant osteocoalescent interface.
Conclusion. This study showed that adding blood can
have different effects on CPCs properties assuming that
they present different setting features. In vivo, it is known
that fibrinogen present in the blood usually polymerize
into fibrin within 12 minutes approximately. Our
hypothesis is as following: when CPC setting time is
longer than 12 minutes, significant modifications, due to
the fibrin polymerization, of textural and mechanical
properties can be observed. On the other hand, for
quicker setting times, those properties are only governed
by apatite crystallization which suggests that fibrinogen is
not able to polymerize into fibrin in that case. Assuming
that biological properties do not seem to be jeopardized
by blood addition, it seems we have found a simple way
to modulated stiffness and plasticity of apatitic CPCs
which could extend applications of these injectable
biomaterials.
Keywords. calcium phosphates cements, blood materials
interaction, textural properties, in vivo responses
Stress (MPa)
Stress (MPa)
LST
13
12
26
22
10
20
9
Blood/SST
18
Blood/LST
8
7
16
14
6
12
5
10
4
8
3
6
2
4
1
0
SST
24
11
2
0,0
0,5
1,0
1,5
2,0
2,5
Distance (mm)
0
0,00
0,25
0,50
0,75
1,00
1,25
1,50
1,75
2,00
Distance (mm)
Fig. 1: Comparative compressive strength tests for CPC
and blood/CPC (A) LST, (B) SST.
(6.O7) A NOVEL EVALUATION OF THE PERFORMANCE OF
SOFT TISSUE REPAIR BIOMATERIALS FOLLOWING
INTRAPERITONEAL IMPLANTATION IN HEALTHY AND
DIABETIC RATS, BY QUANTITATIVE HISTOPATHOLOGY
Alves A (1), Bourges X (2), Yves Bayon (2)
1. Biomatech - Namsa, Chasse / Rhône, France; 2.
Covidien - Sofradim Production, Trévoux, France
Introduction. New generations of biomaterials are facing
the limitations of the ISO standard 10993-6 method of
evaluation, focusing mostly on safety parameters. Their
additional value comes from an improvement of their
performance, eg. by accelerating the wound healing
process with earlier cellularization and neoformation of
tissue, by reducing the foreign body reaction. Specific
histology stainings and methodology were developed for
the quantification of wound healing markers, in healthy
and diabetic rats.
Materials and methods. An abrasion and a surgical defect
were respectively created on the caecum and opposite
peritoneal surface of the Sprague Dawley and diabetic
Zucker rats. The abdominal wall lesion was
intraperitoneally covered with a composite biomaterial
combining a textile and a collagen film. The implantation
time was 21 days post-operatively. Histopathological
evaluations were performed on histological sections
stained with:- SHE & Masson’s Trichrome, for the semiquantitative analysis of selected inflammatory and wound
healing parameters,- Junqueira (collagen polymorphism)
and Feulgen & Rossenbeck stainings (DNA specific) (see
Figure) for the quantitative analysis of the extracellular
matrix, collagen I / III ratio and the cellularization, by
using a customized image analyzer soft ware.
Results/Discussion.
The
semi-quantitative
histopathological analysis showed that the composite
biomaterial tended to yield less signs of inflammation and
fibroplastic tissue formation, in diabetic vs healthy rats.
Differences were more obvious and statistically significant
from quantitative histopathology data: much slower
formation of extracellular matrix, less mature with
predominant collagen III, in diabetic vs healthy rats, as
expected from the literature.
Conclusion. Quantitative histology allows simple and,
more objective and concise evaluations of the
performance of new biomaterials. Its automation should
spare time & money and enable its routine use in this
perspective.
Keywords. quantitative histology, image analysis,
biomaterials, inflammation, wound healing, collagen I &
III, cellularization, in vivo model
(6.O8) CORD BLOOD STEM CELLS EXCEED EMBRYONIC
STEM CELLS IN INDUCING ECTOPIC BONE FORMATION IN
VIVO
Meyer U (1), Handschel J (2), Wiesmann HP (3)
1. MKG Münster; 2. University of Düsseldorf; 3. University
of Dresden
Introduction. Surgery often leads to massive destruction
of the skeleton. Cell-based bone reconstruction therapies
promise new therapeutic opportunities for the repair of
bone. Umbilical cord blood stem cells (USSC) as well as
embryonic stem cells (ESCs) can be differentiated into
osteogenic cells and are a potential cell source for bone
tissue engineering. The purpose of this in vivo study was
to compare these two stem cell lines regarding their
ability to promote ectopic bone formation in vivo.
Methods. Human umbilical cord blood stem cells and
murine ESCs were cultured as monolayer cultures as well
as micromasses and seeded on insoluble collagenous
bone matrix (ICBM). These constructs were implanted in
immundeficient rats. After one week, one, two and three
months CT-scans were performed to detect any
calcifications. Subsequently, the rats were sacrificed and
the implanted constructs were examined histologically.
Results. The radiological examination shows a steep
increase of the mineralised tissue in the USSC-groups.
This increase can be considered as statistical significant
compared to the basic value. Moreover, the volume and
the CaHa-Content were about ten times higher than in
the ESC-group. In contrast, the volume of the
mineralization in the ESC-group increased to a much
lower extend during time and the control-group (ICBM
without cells) almost shows no alterations during the
study. The histological examinations parallel the
radiological findings.
Conclusion. Cord blood stem cells in combination with
ICBM induce ectopic bone formation in vivo stronger than
embryonic stem cells. Thus, this cell population as well as
the biomaterial ICBM might be promising components for
bone tissue engineering.
Keywords. Cord blood stem cells, embryonic stem cells, in
vivo.
(6.O9) INCORPORATION OF INFLAMMATORY SIGNALS IN
BIOMATERIALS MODULATES HUMAN NK CELL BEHAVIOR
LEADING TO IMPROVED MSC RECRUITMENT
Almeida CR (1), Vasconcelos DP (1), Gonçalves RM (1),
Barbosa MA (1)
1. INEB – Instituto de Engenharia Biomédica, Universidade
do Porto, Portugal; Instituto de Ciências Biomédicas
Introduction. Inflammation is one of the first events
taking place upon implantation of a biomaterial. An
exacerbated
inflammatory
response
questions
biomaterial biocompatibility, but on the other hand
inflammation has a central role in regulation of Tissue
Regeneration. Therefore, it may be argued that an “ideal”
inflammatory response is crucial to achieve efficient
tissue repair/regeneration. Natural Killer (NK) cells, being
one of the first populations arriving at an injury site, can
have an important role in regulating bone
repair/regeneration, particularly through interactions
with Mesenchymal Stem Cells (MSCs). Here, we studied
how biomaterials designed to incorporate inflammatory
signals affected human NK cell behaviour and NK – MSC
interactions.
Methods. Ultrathin chitosan films were prepared by
spincoating, to which the pro-inflammatory molecule
Fibrinogen was adsorbed in a monolayer. It was tested
how these films affected peripheral blood NK cells and
bone marrow MSCs behaviour.
Results and conclusions. Adsorption of Fibrinogen to
chitosan films led to a 1.5 fold increase in adhesion of NK
cells, which was accompanied by a change in morphology.
Freshly isolated NK cells were stimulated by MSC to
produce cytokines, but Fibrinogen adsorption did not
affect NK cell IFN-gamma secretion. Most importantly, it
was found that NK cells are capable of stimulating a 3 fold
increase in MSC invasion, a key event taking place in
tissue repair, but did not affect expression of the
differentiation marker ALP, detected by flow cytometry.
Of significant importance, this NK cell-mediated MSC
recruitment was modulated by Fibrinogen adsorption.
Thus, designing novel biomaterials leading to rational
modulation of the inflammatory response is proposed as
a possible route in Tissue Regeneration strategies.
Acknowledgments. Project financed by “COMPETE Programa Operacional Factores de Competitividade”
(FEDER component) and Foundation for Science and
Technology (OE component) – reference PTDC/SAUBEB/099954/2008; and fellowship by Foundation for
Science and Technology (QREN-POPH) – reference
SFRH/BPD/48533/2008.
Keywords.
Tissue
Regeneration,
Inflammation,
biomaterials, NK cells
(6.O10) HEMOCOMPATIBILITY STUDY OF BACTERIAL
CELLULOSE
Andrade FK (1), Silva JP (1), Carvalho M (2), Castanheira
EMS (3), Soares R (4), Gama FM (1)
1. IBB-Institute for Biotechnology and Bioengineering,
Centre of Biological Engineering, University of Minho,
Braga, Portugal; 2. Centre of Thrombosis, Hemostasis and
Vascular Biology, Department of Blood Transfusion and
Blood Bank, Hospital Sao Joao, Oporto, Portugal; 3.
Physics Department University of Minho, Braga, Portugal;
4. Department of Biochemistry (U38 – FCT), Faculty of
Medicine, University of Porto, Oporto, Portugal
Introduction. Vascular grafts must gather various
complex attributes, like good mechanical properties,
post-implantation healing response without any
immunological reaction and no induction of blood
coagulation. Over the years, many strategies were
developed to modify materials for vascular devices. One
strategy involves pre-coating with the tripeptide Arg-GlyAsp (RGD), which improves endothelialization, thus
lowering thrombogenicity. In the present work, the
hemocompatibility of native and RGD-modified bacterial
cellulose (BC) was studied. Despite being a promising
material for vascular replacements, a comprehensive
characterization of the BC-blood interaction, namely in
the presence of RGD peptide, has not been performed to
date.
Methods. Blood from healthy donors was placed in
contact with native or recombinant RGD-treated BC and
parameters related to a material’s hemocompatibility
were determined. These included adsorption of plasma
proteins, clotting times, whole blood coagulation time,
plasma recalcification profiles, platelet adhesion and
hemolysis.
Results. The clotting times (aPTT, PT, FT and PRT) and
whole blood clotting results demonstrate the good
hemocompatibility of BC. A significant amount of plasma
protein adsorbed to BC fibres, presenting albumin a
higher BC affinity than gamma-globulin or fibrinogen.
According to analysis carried out by intrinsic tryptophan
fluorescence, BC-adsorbed plasma proteins tested do not
undergo major conformational modifications. Although
the presence of the adhesion peptide on bare-BC surface
increases the platelet adhesion, when the material was
cultured with human microvascular endothelial cells a
confluent cell layer was readily formed, inhibiting the
adhesion of platelets.
Conclusion. Generally, our data demonstrates that both
native and RGD-modified BCs may be classified as
hemocompatible materials, since they showed to be nonhemolytic and the whole blood coagulation studies show
that the results are comparable to those produced by
currently available materials for blood replacements.
Acknowledgements. Work funded by FCT project
PTDC/EBB-EBI/112170/2009. FKA is supported a CAPES
grant.
JPS
is
supported
by
FCT
grant
SFRH/BPD/64958/2009.
Keywords.
Bacterial
cellulose;
RGD
peptide;
Hemocompatibility; Vascular grafts.
(6.O11) IN VITRO AND IN VIVO BIOCOMPATIBILITY
EVALUATION OF K-CARRAGEENAN HYDROGELS AIMED
AT APLICATIONS IN REGENERATIVE MEDICINE
Popa EG (1), Carvalho PP (1), Dias AF (1), Santo VE (1),
Frias AM (1), Marques AP (1), Dias IR (1), Viegas CAA (1),
Gomes ME (1), Reis RL (1)
1. 3B’s Research Group, Dept. of Polymer Eng., Univ of
Minho, Guimarães, Portugal. IBB Institute for
Biotechnology and Bioengineering, Braga, Portugal.
Introduction. The development of biomaterials for
biomedical applications always requires extensive
biological testing to demonstrate the safety of both the
material and its degradation components. K-carrageenan
is a naturally occurring polysaccharide which forms a
hydrogel with potassium ions and the temperatureinduced gelation enables its application as an in vitro cellcarrier or as an in vivo injectable system. The aim of this
study was to evaluate these novel systems as
biomaterials by in vitro biological screening and by in vivo
implantation to assess for the inflammatory response.
Methods. In vitro evaluation: The cytotoxicity of the
hydrogels was evaluated under standard tests using the
L929 cell. Furthermore, the viability and proliferation of
encapsulated human adipose stem cells (hASCs) was
determined by fluorescence staining and DNA
quantification. In vivo evaluation: discs of k-carrageenan
were subcutaneous implanted in a wistar rats for up to 15
days. The materials (agarose-control material and kcarrageenan) were positioned into each pocket. Control
animals with empty defect and empty defect injected
with lipopolysaccharide were used. After each time
period, the biomaterial, surrounding tissue and nearby
lymph nodes were explanted and used for histological
analysis and molecular biology evaluation.
Results. The cytotoxicity test and biological evaluation of
k-carrageenan revealed that this polymer is not cytotoxic
and enables long term viability and proliferation of cells in
vitro. At implant retrieval there were no macroscopic
signs of a considerable inflammatory reaction in any of
the animals and no cellular exudates was formed around
the implants.
Conclusions. The results indicated that k-carrageenan is a
biocompatible material and does not cause a severe host
response. These results together with those obtained
regarding the properties of k-carragenan investigated
under other studies indicate that theses hydrogels may
be successfully applied in tissue engineering approaches.
Acknowledgements. The authors gratefully acknowledge
Portuguese Foundation for Science and Technology (FCT)
for the PhD grants of Popa EG (SFRH/BD/64070/2009),
Carvalho P.P. (SFRH/BD/44128/2008), Santo VE (SFRH /
BD / 39486 / 2007) and Frias AM (SFRH
/BPD/45206/2008).
Keywords. hydrogels, biomaterials, in vitro response,
subcutaneous implantation, inflammatory response,
cartilage tissue engineering
Stress (MPa)
Stress (MPa)
LST
13
12
26
22
10
20
9
Blood/SS
18
Blood/LS
8
7
16
14
6
12
5
10
4
8
3
6
2
4
1
0
SST
24
11
2
0,0
0,5
1,0
1,5
2,0
2,5
Distance (mm)
0
0,00
0,25
0,50
0,75
1,00
1,25
1,50
1,75
2,00
Distance (mm)
Fig. 1: Comparative compressive strength tests for CPC
and blood/CPC (A) LST, (B) SST.
(6.O12) HUMAN HAIR KERATINS FOR TISSUE
ENGINEERING
Ng KW (1), Wang S (1), Taraballi F (1)
1. School of Materials Science & Engineering; Nanyang
Technological University, Singapore
Introduction. Natural materials, including proteins and
polysaccharides, are being widely used as scaffolds for
tissue engineering because they provide a bioactive
platform for cellular processes and more closely resemble
the native in vivo environment. However natural
materials, derived mostly from animal sources, present
risks of pathogen transfer and may be limited in
abundance. This project was initiated to explore the
feasibility of transforming human hair, one of the largest
sources of bio-waste we produce, into templates that
could be used in biomedical applications. Hair is attractive
because 1) it is readily available, 2) it is a rich source of
keratins which have the ability to self-assemble into a
matrix and contain cell adhesion motifs, 3) it offers the
possibility to produce autologous scaffolds for clinical
applications.
Materials Methods. Keratins were extracted from
random human hair samples in reducing conditions, using
protocols modified from reported literature. Samples
were characterised by Western Blotting to evaluate the
profile of keratins obtained. Keratins in solution were
subsequently processed into gels, freeze-dried discs or
fibrous foams for subsequent feasibility studies.
Preliminary cell culture studies were conducted to
evaluate in vitro biocompatibility.
Results. In agreement with the literature, we
demonstrated that keratins can be effectively extracted
from human hair samples. Keratin gels, freeze-dried discs
and fibrous foams were successfully fabricated. Scanning
electron
microscopy
images
show
that
3D,
interconnected micro-porous architectures can be
produced within the freeze-dried discs and fibrous foams,
characteristic of scaffolds suitable for use in tissue
engineering. Preliminary cell culture studies show that
the keratin templates produced can support cellular
attachment and proliferation.
Conclusions. We showed that keratins extracted from
human hair have the potential to be processed into
various templates. Future studies will focus on using
these in specific tissue engineering applications.
Acknowledgements. This work is funded by the National
Medical Research Council (NIG09may016).
(6.P1) POLYMER SURFACES COATED WITH HYDROGEL TO
IMPROVE BLOOD COMPATIBILITY
Butruk B (1), Ciach T (1)
1. Warsaw University of Technology, Faculty of Chemical
and Process Engineering, Warsaw, Poland.
Introduction. The aim of presented research was to
develop a method for manufacturing hemocompatible
coatings for blood-contacting devices. We present a
simple method for fabrication of hydrogel coatings for
cardiovascular devices. Polyvinylpyrrolidone (PVP) was
chosen as a hydrophilic polymer to produce hydrogel
network due to its highly biocompatibility and wide
applications in medicine.
Methods. Hydrogel coatings of polyurethane (in a form of
discs) were fabricated in a two-step method. First, the PU
discs were immersed in a solution containing given
amounts of crosslinking agent (EGDMA) and cumene
hydroperoxide for 15 minutes at 25°C. After that time,
samples were placed in a water solution containing given
amounts of PVP, FeCl2 and ascorbic acid for 15 minutes at
25°C. Polymer discs were then washed and dried.
Blood-biomaterial interactions were evaluated using a
platelet analyzer (Impact-R, DiaMed). A given volume of a
whole-blood sample was dropped onto the characterized
surfaces and shear stress was applied to simulate arterial
flow conditions. The platelet consumption was calculated
as a difference between the initial number of platelets
present in blood sample and the number of platelets after
the test.
Results. Presented method is based on free-radical
macromolecular polymerization. Cumene hydroperoxide
is a source of radicals produced in the redox reaction with
Fe2+ ions. Macroradicals recombination leads to PU-PVP
grafting, PVP crosslinking and hydrogel formation. The
results showed that the platelet consumption decreased
from 56% (for unmodified PU) to 10% (for PU grafted with
PVP).
Conclusion.
Polyurethane
grafted
with
polyvinylopyrrolidone seems to be promising material for
cardiovascular applications. Hydrogel coating greatly
reduced the level of platelet adhesion and activation.
Acknowledgments. This work has been supported by the
Polish Artificial Heart Project and the European Union in
the framework of European Social Fund through the
Warsaw University of Technology Development
Programme.
Keywords. hydrogel; surface modification; blood
compatibility
(6.P2) IN VIVO FOREIGN BODY REACTION TO
MICROSPHERES COMPOSED OF BIODEGRADABLE
HYDROPHILIC MULTI-BLOCK COPOLYMERS
Zandstra J (1), Zuidema J (2), Dimitropoulou D (1),
Hiemstra C (2), Steendam R (2), Popa ER (1)
1. University Medical Center Groningen; 2. InnoCore
Technologies BV
introduction. Biodegradable hydrophilic multi-block
copolymers composed of polyethyleneglycol and Lactide
(PEG/LA-MBCP) are considered promising materials for
the preparation of controlled release microsphere (MSP)
formulations for site-specific or systemic drug delivery. To
determine the in vivo biocompatibility of this new class of
materials we examined the foreign body reaction (FBR) to
subcutaneously administered PEG/LA-MBCP MSPs.
Furthermore we studied the FBR in relation to particle
size to determine the most optimal size of PEG/LA-MBCPbased MSP for use as injectable drug delivery depots.
Experimental Methods. MSP (particle size 5-200µm)
were prepared by a standard W/O single emulsion
process. Lyophilized MSP were suspended in sterile water
containing 0.4% sodium carboxymethylcellulose and
injected subcutaneously on the back of F344 rats. MSP
and surrounding tissue were retrieved after 7 days.
General histology was evaluated by toluidin blue staining.
The FBR was studied by staining for macrophages (ED-1),
fibroblasts (FSP-1) and potential wound healing
macrophages (ED-1/FSP-1).
Results. A very mild FBR to PEG/LA-MBCP MSP was
observed, as indicated by the absence of a fibrous capsule
around the MSP. Large (50-200µm) MSP were surrounded
by 1-2 cell layers of macrophages. A moderate
macrophage infiltration was present between the
microspheres and was interspersed with occasional
fibroblasts and ED-1+/FSP-1+ macrophages. Small (<µm)
MSP were phagocytised while large ( 30>50-200µm) MSP
occasionally elicited giant cell formation.
Conclusion. PEG/LA-MBCP MSP demonstrated excellent
in vivo biocompatibility. Small PEG/LA-MBCP MSPs were
preferentially phagocytised, while larger MSPs were not.
It is concluded that this new class of biodegradable
hydrophilic polymers provides a suitable platform for
parenteral drug delivery and that microspheres of 20 – 50
micron should preferably be used to minimize the overall
foreign body reaction.
Acknowledgements. This research forms part of the
Project P3.02 DESIRE of the research program of the
BioMedical Materials institute, co-funded by the Dutch
Ministry of Economic Affairs.
Keywords. biocompatibility, controlled release, foreign
body reaction
(6.P3)
ELASTIN
MICROSPHERES
ARE
THROMBORESISTANT BUT NOT IMMUNORESISTANT
Fitzgerald KT (1), Srokowski EM (2), Woodhouse KA (3),
Gallagher WM (1).
1. UCD School of Biomolecular and Biomedical Science,
UCD Conway Institute, University College Dublin, Belfield,
Dublin 4, Ireland; 2. Chemical Engineering and Applied
Chemistry, Institute of Biomaterials and Biomedical
Engineering, University of Toronto, Toronto Ontario,
Canada; 3. Chemical Engineeing, Queen’s University,
Kingston Ontario, Canada.
Introduction. Finding an appropriate microsphere that
can be systemically administered without causing
thrombosis or an immune response is crucial to the
success of cardiovascular therapy and diagnosis. Recent
work [1,2] has shown that a family of recombinant human
elastin-like polypeptides (ELPs) has the potential to
protect against thrombotic events. Herein we investigate
the effects of platelet and immune cell activation to ELPs
(ELP1 and ELP4) that differ by molecular weight and
sequence length.
Materials and Methods. Whole blood collected in sodium
citrated tubes was tested for platelet activation (CD61
and CD62P) and leukocyte activation (CD62L, CD45 and
CD11b) and analyzed using flow cytometry. Physiological
blood flow (13.5 dyne/cm2) was simulated using Cellix’s
microfluidic platform. The biochip channels (400 x 100
µm) were coated with collagen, ELPs and uncoated
control channel.
Results. Preliminary results showed that shear stress
induced platelet activation (non-coated channel). Blood
subjected to collagen-coated channels resulted in a 50%
increase of P-selectin expression. Both ELP1 and 4
showed thromboresistant effects (Fig. 1). Initial results of
the immunoresistant effects of ELPs indicate that CD11b
was not impacted by the presence of the ELPs (data not
shown).
Discussion and Conclusions. These preliminary results
illustrate that the ELPs have thromboresistant properties.
However, the mechanism of this protective effect has to
be
further
studied.
Interestingly,
the
ELP
thromboresistant properties appear to be platelet
specific, clearly not extending to the leukocyte
population.
References. [1] Woodhouse et al. Biomaterials, 25, 4543
(2004). [2] Srokowski et al. J Biomat Sci Polym Ed, (2010)
(accepted - JBS3083).
Acknowledgments. This work is supported by the Science
Foundation Ireland under Grant No. 07/SRC/B1163. The
Conway Institute is funded by the Programme for Third
Level Institutions (PRTLI), as administered by the Higher
Education Authority (HEA) of Ireland. We acknowledge
funding support by the Canadian Institutes of Health
Research.
Keywords. elastin, cardiovascular, platelet
% Expression of P-selectin on Platelet Population
% P-selectin Expression
100
75
50
25
ELP4
ELP1
Noncoated
Collagen
0
(Treatments)
Fig. 1. Expression of CD61/CD62P on blood samples that
were subjected to shear stress (13.5 dyne/cm2) over
collagen, ELP1, ELP4 and non-coated channels.
(6.P4) HISTOCHEMICAL EVALUATION OF IN VIVO
BIOCOMPATIBILITY OF MODIFIED CARBON FIBRES
Menaszek E (1), Rajzer I (2)
1. Departament of Cytobiology, Collegium Medicum, UJ
Jagiellonian University, Poland; 2. Institute of Textile
Engineering and Polymer Materials, ATH University of
Bielsko-Biala, Poland
Carbon fibres offer an unusual potential in designing new
biomaterials for medical applications. They were
attempted to use in the reconstruction of fibrous tissue
and for the repair of cartilage and bone defects. The
objective of our study was to investigate the
biocompatibility of carbon fibres: porous, and coated with
hyaluronic acid.
Methods. Three types of carbon fibres: non-modified
(CF), porous (CFP), and modified with hyaluronic acid
(CF/HA) were prepared from polyacrylonitrile precursor.
The in vivo studies were carried out using the rat soft
tissues as a model. Equal portions of CF, CFP, and CF/HA
carbon fibres were implanted into the skeletal muscle of
rats. After 1, 4, 12, and 30 weeks from the surgery, the
implants along with their surrounding tissue were
excised, frozen in liquid nitrogen and cut in a cryostat
microtome. The obtained slides were investigated
through histological and histochemical methods to
estimate the intensity of inflammation, production of
collagen, and metabolic activity of tissues surrounding the
implant.
Results. The activity of mitochondrial oxidative enzymes:
cytochrome c oxidase and NADH dehydrogenase in the
muscle fibres in close proximity to the implants was only
slightly lower than in those further away. The presence of
a foreign body (i.e. carbon fibres) evoked a prolonged
inflammation response (especially around CFP), still
intense even in the 30-week series. On the other hand,
inflammatory cells helped in the process of regeneration
and prevented the formation of a connective fibrous
capsule. The fibrous capsule around CFP and CF/HA
implants was thin or not present at all - the fibres were in
direct contact with the muscle tissue.
Conclusion. The regeneration and enzymic activity of
muscle tissue together with the lack of fibrous capsule
suggest that the carbon fibres used in our study are
biocompatibile and are suitable as scaffolds for tissue
engineering.
Keywords. carbon fibres, histochemistry, biocompatibility
(6.P5) NEW MICROSCOPY APPROACH TO EXAMINE THE
HOST TISSUE INCORPORATION OF DIFFERENT
BIOPROSTHESES USED FOR ABDOMINAL HERNIA REPAIR
Pascual G (1), Rodríguez M (2), Sotomayor S (2), Moraleda
E (2), Bellón JM (2)
1. Department of Medical Specialities, Faculty of
Medicine, University of Alcala, Alcalá de Henares, Madrid,
Spain. (CIBER-BNN); 2. Department of Surgery, Faculty of
Medicine, University of Alcala, Alcalá de Henares, Madrid,
Spain. (CIBER-BNN).
Introduction. Biological prosthetic materials have been
added to the materials available for the repair of hernial
defects in the abdominal wall. Such materials share the
important feature that they are gradually degraded in the
host resulting in the formation in its place of a neotissue,
which in the long term will completely replace the
biomaterial.
We assessed the host tissue incorportation of different
bioprostheses, using a new tool that combines
immunofluorescence confocal microscopy technique with
differential interference contrast (DIC), making it possible
to distinguish newly formed collagen from that of the
bioprosthesis.
Methods. Partial hernial defects were created in the
abdominal wall of rabbits and repaired using crosslinked
(Permacol®(Pe),collamend®(Coll)) and non-crosslinked
(Surgisis®(SIS)) bioprostheses. Eptfe (Preclude®) was used
as control. 14, 30, 90 and 180 days post-implant,
specimens
were
taken
for
microscopy,
immunohistochemistry and qpcr to determine host tissue
ingrowth and collagen I/III gene and protein expression.
Results. Eptfe was encapsulated by neoformed tissue
while bioprostheses became gradually infiltrated by host
tissue. SIS showed better tissue ingrowth and was more
rapidly degraded. 14/30 days after placement, the
different bioprotheses showed sparse or no
immunostaining for collagen I. The levels of this protein
increased over time, showing at 90/180 days their
staining peak. In the SIS, staining was more discrete and
evenly distributed throughout the biomaterial’s thickness.
At 14 days postimplant, collagen III was highly expressed
in the neoformed tissue, and this expression rose at 30
days and continued increasing in the long term. At 14
days, Pe and Coll induced upregulated collagen 1 and 3
gene expression, while SIS only showed increased
immature collagen III expression at 90 days.
Conclusions. This new microscopy approach allows
monitoring the process of tissue integration and
bioprosthesis degradation showing that despite the
crosslinked collagen bioprostheses promoting less tissue
ingrowth than SIS, they became gradually replaced by
good quality host tissue.
This study was supported by a grant from the Fundación
Mutua Madrileña 2008 (FMM08), Madrid, Spain.
Keywords. collagen bioprostheses, abdominal hernia
repair, tissue integration.
(6.P6)
BIOMATERIALS-ASSOCIATED
INFECTIONS.
INFLUENCE OF PROPHYLACTIC ANTIBIOTICS ON THE
COMPETITION BETWEEN BACTERIA AND MAMMALIAN
CELLS FOR THE BIOMATERIAL SURFACE
Aleyt T (1), Subbiahdoss G (1), Kuijer R (1), van der Mei HC
(1), Busscher HJ (1)
1. University Medical Center Groningen
Introduction.
Biomaterials-associated
infections
represent a major clinical problem. The fate of a
biomaterial implant has been described as a ‘race for the
surface’ between microorganisms and tissue cells.
Microorganisms are frequently introduced on an implant
surface during surgery giving them a head start in the
race for the surface. The aim of this study was to assess
the influence of one shot of prophylactic antibiotics on
the competition between bacteria and mammalian cells
in an in vitro model.
Materials and Methods. The model comprised
Staphylococcus aureus (ATCC 12600) and U2OS
osteoblast-like cells cultured together on PMMA surfaces
in a parallel plate flow cell in a modified culture medium
(MCM) for up to 72 h. S. aureus were deposited on the
surface followed by seeding of U2OS cells. Then flow was
started with MCM containing Cephtolin (1xMBC) for 8 h.
Subsequently, flow was switched to MCM only for the
additional 64 hours. Biofilm growth was assessed using
live-dead staining. U2OS cell number and morphology
were measured after staining with phalloidin, CLSM and
image analysis at 1.5 and 72 h.
Results. In the absence of antibiotics, U2OS cells died
within 24 h in the presence of adhering S. aureus. In the
presence of Cephtolin, no change in U2OS cell
morphology was observed compared to control (U2OS
cells without S. aureus). A slow growth of S. aureus
biofilm was observed after 8 h of antibiotic treatment.
The number of U2OS cells at 72 h was significantly
reduced compared to control.
Conclusions. One shot of Cephtolin did not kill all
bacteria. The slow growth of S. aureus after incubation
with antibiotics is suggestive for decreased metabolic
activity. Longer term antibiotic treatment should clinically
be considered.
Keywords. infection; biomaterial-associated infection;
antibiotic
7. BIOREACTORS TECHNOLOGIES FOR
TISSUE ENGINEERING
Chair: Aaron Goldstein
Co-chair: Smadar Cohen
Keynote speaker: Fergal O'Brien
Organizer: Aaron Goldstein
Synopsis: Cell based approaches in regenerative medicine
frequently rely on in vitro conditioning of cells to permit
their proliferation, differentiation, and organization into
tissue-like structures with functional properties
approaching those of normal tissue. During this period of
conditioning, bioreactors can be used to exert external
stimuli (e.g., mechanic strain, hydrodynamic pressure and
shear, electrical fields) that facilitate matrix deposition
and tissue organization, and improve quantitative
measures of tissue function.
In this proposed symposium for the TERMIS-EU meeting
in 2011, we would include research presentations that
describe the development and testing of bioreactors for
regeneration of a large range of musculoskeletal and
cardiovascular tissues. This includes (but is not limited to)
bone, cartilage, tendon/ligament, skeletal muscle, cardiac
muscle, blood vessels, and heart valves. Presentations
concerning fundamental studies of bioreactor
performance, basic studies of cell/tissue development in
vitro, and translation studies of tissue efficacy in vivo
would be encouraged. Special emphasis will be placed on
presentations describing cutting-edge research, such as
bioreactors designed to mimic the dynamic
physical/mechanical stimuli that exist in vivo and
strategies that employ multiple cell types.
(7.KP) COLLAGEN-BASED SCAFFOLDS IN TISSUE
ENGINEERING: APPLIED BIOMATERIALS AND CELLULAR
RESPONSE TO FLOW PERFUSION
O’Brien FJ (1)
1. Dept. of Anatomy, Royal College of Surgeons in Ireland
& Centre for Bioengineering, Trinity College Dublin
Tissue engineering uses a combination of: (i) biomaterial
scaffold (ii) cells and (iii) signalling mechanisms (such as
growth factors or mechanical stimuli) to restore the
function of damaged or degenerated tissue in vivo or to
culture tissue in vitro which can be used for implantation.
Recent work in our laboratory has developed a series of
collagen-based scaffolds with the optimal composition,
pore structure and stiffness to promote bone formation
in vitro and healing in vivo. In the cellular area, we are
investigating the osteogenic, chondrogenic and
angiogenic potential of mesenchymal stem cells on these
scaffolds and we have a particular interest in using
biophysical stimuli to regulate stem cell differentiation. In
this area, we have developed a flow perfusion bioreactor
system and have shown that flow perfusion increases the
osteogenic potential of cells seeded on the scaffolds and
quantified resultant cellular shear stresses using a
computational fluid dynamics (CFD) model. Our results
demonstrate that mechanism of cellular attachment in
the scaffolds is critically important in regulating the
optimal biophysical stimuli required to enhance
osteogenic potential. We have shown that wall shear
stresses required to activate an osteogenic response in
calcium phosphate scaffolds with large pores are
approximately 40 times higher than in collagen-GAG
scaffolds with small pores. Furthermore, the results
suggest that levels of cellular deformation are as
important as cellular shear stress in regulating
differentiation following flow. In scaffolds with smaller
pores, cells can bridge the pores, which exposes them to
greater deformation, allowing for enhanced osteogenic
response at flow rates insufficient to promote a response
in scaffolds with larger pores. Using results from the CFD
model, we have been able to determine the requisite
stimuli required for longer term bioreactor culture and
have demonstrated the potential of using the system to
improve cell distribution and enhance osteogenesis.
Keywords. collagen-based scaffold, flow perfusion,
computational fluid dynamics, cell distribution,
osteogenesis
(7.O1) MICROBIOREACTORS FOR CARDIAC TISSUE
ENGINEERING
Xiao Y (1), Thavandiran N (1), Au H (1), Radisic M (1)
1. University of Toronto
In contractile tissues such as myocardium, functional
properties are directly related to cellular orientation and
elongation. Thus, tissue engineering of functional cardiac
patches critically depends on our understanding of the
interaction between multiple guidance cues such as
topographical, adhesive and electrical cues.
One of our goals was to determine the interactive effects
of contact guidance and electrical field stimulation on
elongation and orientation of cardiomyocytes and
fibroblasts, major cell populations of the myocardium.
We developed a precise microfabricated system,
incorporating topographical and electrical cues on a
single chip. The cell culture chips were created by hot
embossing of polystyrene, with microgrooves and
microridges of precisely defined depth, width and
periodicity.
The
two
gold
electrodes
were
electrodeposited 1cm apart such that the microgrooves in
between were oriented either parallel or perpendicular to
the electrodes. Importantly, simultaneous application of
biphasic electrical pulses and topographical cues resulted
in gap junctions confined mainly at the cell-cell ends
rather than the punctuate distribution normally found in
neonatal cells. Overall, we observed that i) cardiomyocyte
and fibroblast elongation on smooth surfaces was
significantly enhanced by electrical field stimulation and
ii) topographical cues were a significantly stronger
determinant of cardiomyocyte orientation than the
electrical field stimulation. The orientation and
elongation response of cardiomyocytes was completely
abolished by inhibition of actin polymerization and only
partially by inhibition of phosphatidyl-inositol 3 kinase
pathway.
Our current efforts focus on development of a microarray
of cardiac organoids for drug and cell testing, where
tissues are created by self-organization of embryonic
stem cell derived cardiomyocytes around two microposts.
Additionally, to create biological wires of 1-10cm scale
capable of propagating electrical impulses, we employ
self organization of cardiomyocytes around sutures
placed in microbioreactor wells. Thus, the three
microbioreactor configurations we developed provide
control of cellular microenvironment to enable
engineering of functional cardiac organoids.
Keywords.
Bioreactor,
microenvironment,
cardiomyocyte, electrical stimulation
(7.O2) NEW GENERATION BIOREACTOR FOR IN VITRO
ENGINEERING OF TUBULAR STRUCTURES
Asnaghi MA (1), Stefani I (1), Mantero S (1)
1. Politecnico di Milano, Department of Bioengineering
Introduction. The clinical need to replace tubular organs
with functional substitutes, where conventional
reconstruction techniques are inadequate, is growing
exponentially. Recently, there has been a growing
optimism that cell-based tissue engineering methods may
provide effective solutions and early promising results
have been reported. We have already shown in a clinical
setting that our previously developed double-chamber
rotating bioreactor allowed multiple cell types to be
grown onto a decellularized trachea [1,2]. Here we
introduce a second-generation bioreactor for tubular
construct engineering with improved functionalities.
Methods. Major aims of the bioreactor design were to:
allow proper seeding and culturing of different cell types
on both sides of a tubular matrix, promote efficient mass
transport within a construct of clinically relevant
dimensions and stimulate cells with hydrodynamic
stimuli. Modularity, optimization of assembly procedures,
control and automation over the entire process were
further key requirements.
Results. Our bioreactor combines scaffold pre-tensioning,
rotation and luminal perfusion, exposing cells
alternatively to liquid and gas phases if half immersed in
culture medium. A novel apparatus for the automatic
medium exchange was also realised and coupled to the
bioreactor, significantly contributing to minimize
contamination risks and to protect homeostasis of the
culture milieu. The manufactured system was benchtested under different operating conditions, and
preliminary cell culture trials were performed with
positive outcome: higher cell survival and much better
colonisation throughout the scaffolds thickness with
respect to static controls were obtained.
Conclusions. The improved bioreactor is an effective and
versatile system that could be used with different
scaffolds (Ø, L) to in vitro engineer tubular structures,
e.g., trachea and blood vessels. Based on the collected
promising results, we have been in further experimental
sessions to better investigate its role in driving cell
response.
Acknowledgements: Supported by Regione Lombardia
and by Harvard Bioscience, Inc. through a sponsored
research agreement.
References.
1. Macchiarini P, Jungebluth P, Go T, Asnaghi MA, et al.,
Lancet 372:2023-30, 2008.
2. Asnaghi MA, Jungebluth P, Raimondi MT, Dickinson SC,
et al., Biomaterials 30(29):5260-9, 2009.
Keywords. bioreactor, tubular structures, enabling
technologies
(7.O3) AUTOMATED, ONLINE, REAL-TIME MONITORING
OF CULTURE PARAMETERS IN MULTIPLE INDEPENDENT
CHAMBERS OF A PERFUSION BIOREACTOR
Turrisi C (1), Talò G (2), Arrigoni C (3), Moretti M (2,3)
1. SKE Advanced Therapies S.r.l., Milano, Italy;
Bioengineering Department, Politecnico di Milano,
Milano, Italy; 2. I.R.C.C.S. Galeazzi Orthopedic Institute,
Milano, Italy; 3. GSD Foundation, Cell and Tissue
Engineering Lab, Milano, Italy
Introduction. Perfusion bioreactors represent a promising
possibility for the development of automated,
standardized, cost-effective, and safe manufacturing
processes of engineered tissue substitutes.
Based on a previously developed perfusion bioreactor for
seeding and culture of cell-scaffold constructs (NASA
techbrief), in this study we developed and tested an
automated device for online, real-time monitoring of
critical culture parameters.
Materials and Methods. The bioreactor has been
equipped with a motor driven automated sensing system
(Fig.1a) and with a customized software (Fig.1b) able to
monitor up to 18 independent culture chambers.
Validation tests were performed to monitor pH value
within buffers and non pre-equilibrated culture medium
without cells, with reference to induced environmental
changes monitoring capability.
Moreover, expanded primary human articular
chondrocytes were seeded and cultured on collagen
(UltraFoam) scaffolds for 7 days in DMEM+10%FBS, with
different cell densities. pH and pO2 were optically
monitored and ΔpH (difference between pH upstream
and downstream the scaffold) was calculated for each
chamber. Inoculation of bacteria was performed, so as to
simulate possible contamination and detect related
changes in pH or pO2.
Results and Discussion. The sensing system was able to
detect induced environmental changes due to incubator
door opening, medium change and accidental blackout, in
non pre-equilibrated DMEM+10%FBS, without cells.
Online, real-time parameters monitoring enabled
observation of progressive pH drop during cell dynamic
culture and of sudden drop both in pH and pO2 due to
induced bacterial contamination.
The different cell densities, estimated by DNA and MTT
assays at the end of the experiment, could also be
discriminated by different ΔpH values, detected by the
system, thus giving an index of culture progression,
assessable in real-time.
Conclusions. Our perfusion bioreactor, with automated,
online monitoring of culture parameters, can represent a
step forward towards a reliable device for the safe and
automated manufacturing of biological tissues.
Keywords. perfusion bioreactor, online monitoring,
automation
Figure 1: a) The sensorized bioreator system; b) Front
panel of the cusutomized software.
(7.O4) MODELING OF FLOW-INDUCED SHEAR STRESS
APPLIED ON 3D CELLULAR POROUS SCAFFOLDS
Lesman A (1), Blinder Y (1), Levenberg S (1)
1. Department of Bio-Medical Engineering, Technion Israel Institute of Technology, Haifa, Israel
Introduction. Novel tissue engineering bioreactor
systems are designed to overcome the size limitations of
engineered tissue, which are dictated by oxygen and
nutrient diffusion rates. Our bioreactor system employs
direct perfusion through porous biopolymer scaffolds,
which is meant to simulate physiological interstitial flow
conditions. In order to properly estimate the flowinduced shear stress to which the cells are exposed, a
computational fluid dynamics (CFD) model was
developed. This model takes into account the complex 3D
structure of the porous biopolymer scaffold and the
growth of the cell layer and calculates the shear stress
distribution as a function of the controllable flow rate and
culturing time.
Goals. Develop a CFD model to estimate flow-induced
shear stress applied on cells seeded on a porous
biopolymer scaffold in a direct perfusion bioreactor, as a
function of inflow rate and growing tissue layer thickness.
The current model was designed to predict high shear
stress values within the physiological range naturally
sensed by vascular cells (1–10 dyne/cm^2).
Results. Representational maps of velocity and 3D shear
stress distribution were obtained for each of the models.
Analysis of the calculated wall shear-stress distribution in
the acellular scaffold model shows that while the shear
stress values positively correlated with increasing inflow
velocities, the distribution pattern remained largely
unvaried. As is expected in low Reynolds flow (Re<0.02),
the flow regime, while convoluted, was absolutely
laminar, and mean shear-stress remained proportional to
the inlet velocity.
Conclusions. Our model provides an estimation of the
dynamic microenvironment to which cells are exposed in
our direct perfusion bioreactor. As such, it represents a
useful tool for perfusion bioreactor system design, and
provides an added level of control over experimental
setups.
Keywords. bioreactor, CFD, shear stress
(7.O5) A NEW SEEDING AND CONDITIONING
BIOREACTOR FOR HEART VALVE TISSUE ENGINEERING
Akra B (1), Koenig F (2), Haas U (1), Thierfelder N (1),
Aleksieva G (1), Pfeifer S (2), Wintermantel E (2), Bombien
R (1), Schmitz C (1), Reichart B (1)
1. Department of Cardiac Surgery, Laboratory for Tissue
Engineering, Grosshadern Medical Center, LudwigMaximilian-University, Munich, Germany; 2. Chair of
Medical Engineering, Technical University Munich,
Garching, Germany
Introduction. The purpose of the study was to develop a
new seeding and conditioning bioreactor that permits the
application of an endoscope for online monitoring and
documentation.
Methods. A new system was designed to provide a low
pulsatile flow that grants the correct opening and closing
of the valve leaflets without high shear stresses. This
system consists of three main elements, the actuation
unit, the core unit and the monitoring unit. The actuation
unit generates an accurately adjustable pulsatile flow in
the core unit. The core unit holds the heart valve and
ensures a circulating flow through the valve to achieve an
opening and closing of the valve leaflets. The monitoring
unit fixates an endoscope for a precise monitoring of the
valve leaflets.
Results. Bioreactor permitted an effective and sterile
valve conditioning and/or seeding. It allowed both
recording and documentation of the valve performance
under pulsatile flow conditions. Microbiological tests of
cell medium after 5 days conditioning revealed no
bacterial contamination.
Conclusions. New bioreactor offers a new method that
allows colonized cells to adapt to shear stress and to
establish a strong extracellular matrix.
Keywords. Bioreactor; Heart valve; Seeding; Conditioning
(7.O6) DESIGN OF A FLOW PERFUSION BIOREACTOR FOR
LONGITUDINAL
MONITORING
OF
MINERALIZED
EXTRACELLULAR MATRIX GROWTH
Hofmann S (1), Wechsler O (1), Vetsch J (1), Müller R (1)
1. Institute for Biomechanics, ETH Zurich, Zurich,
Switzerland
Introduction. Bioreactors are widely applied to create
controlled in vitro conditions that mimic the natural
environment engineered tissues. Mechanical stress
through flow perfusion as well as through improved
nutrient transport have been shown to improve cell
seeding efficiency, cell proliferation and differentiation
into bone-like tissue. Non-destructive micro-computed
tomography has been shown to provide qualitative and
quantitative 3D data on mineralized extracellular matrix
(ECM) development both in end-point measurements as
well as in longitudinal monitoring studies. We sought to
design bioreactors that combine the two techniques in
order to follow the reaction to mechanical stimuli of each
sample individually in a controlled environment.
Methods. Custom-made bioreactors were designed to
fulfill the demand for sterile conditions during
measurements and medium exchange, radio-opacity, and
at the same time controllable fluid flow patterns.
Mesenchymal stem cells were seeded onto disk-shaped
porous silk fibroin scaffolds of 8 mm diameter and 2-3
mm height and cultured under static and dynamic (0.2
ml/min) conditions in osteogenic medium for 7 weeks.
Results. The device offers a cartridge-chamber system
that allows investigating into 24 stiff and/or compliant
scaffolds in parallel and provided sterility throughout the
whole culture time, during micro-CT scanning and media
exchange. While the control group showed increasing
mineralized ECM, the application of a perfusion flow of
0.2 ml/min resulted in increased cell proliferation without
cell differentiation but a better cell distribution
throughout the scaffold volume.
Conclusions. Longitudinal monitoring studies over several
weeks can be performed with this new bioreactor design
without contamination. Optimal dynamic parameter
settings still have to be determined to maximize
mineralized ECM content. Additionally, this bioreactor
may improve current seeding strategies through better
distribution of cells throughout the scaffold volume.
Acknowledgments: We would like to acknowledge
funding from the RMS Foundation, Bettlach, Switzerland.
Silk was kindly provided by Trudel Silk Inc., Zürich,
Switzerland.
Keywords. flow perfusion, bioreactor, monitoring, stem
cells
(7.O7) CYCLIC HYDROSTATIC FORCE APPLIED IN A
CUSTOM BIOREACTOR STIMULATES ENHANCED BONE
DEVELOPMENT IN THE FOETAL CHICK FEMUR IN VITRO
Henstock JR (1), El Haj AJ (1)
1. Institute of Science and Technology in Medicine, Keele
University, UK
Introduction. Hydrostatic force has been suggested as an
important stimulus by which osteochondral and
progenitor cells sense and respond to mechanical loading
in vivo. A model system has here been established to
investigate bone development in the chick foetal femur
ex vivo in response to low levels of applied cyclic
hydrostatic forces using a custom designed bioreactor
(Tissue Growth Technologies).
Methods. Femurs isolated from day 11 chick embryos
were cultured in vitro in alpha or osteogenic medium. A
regime of one hour stimulation per day at 1 Hz, cycling
between 0 – 40 PSI (276 MPa) was applied under
standard cell culture conditions for 10 out of 16 days. End
point analysis was by µCT and Alizarin red assay for
matrix mineralisation.
Results. After 16 days femurs in both osteogenic and
alpha media that received stimulation were visibly more
compact than unstimulated femurs in alpha media. µCT
analysis revealed a significant increase in the density and
volume of the bone collars in osteogenic media with
stimulation over unstimulated controls (fig. 1.). A smaller
effect was observed for stimulated femurs in alpha
medium. All stimulated femurs displayed increased bone
collar density regardless of media type – this was
supported by a calcium assay which showed that similar
amounts of calcium were present in all stimulated
femurs, approximately an 8-fold increase in calcium over
unstimulated controls in alpha medium.
Conclusions. Cyclic hydrostatic force stimulates bone
collar growth and mineralisation in the chick femur ex
vivo. Increased bone formation was observed in both
media types, indicating that this type of stimulation can
independently stimulate osteogenesis and also act
synergistically with soluble factors to enhance bone
development in vitro. This stimulation regime could
therefore be applied to cell-seeded 3D scaffolds for in
vitro conditioning prior to their implantation for
applications in osteochondral tissue engineering.
Comments. Fig. 1. µCT of bone collars from chick femurs
cultured in alpha or osteogenic media +/- cyclic
hydrostatic stimulation.
Keywords. Bioreactor, Osteogenesis, Mechanical
Stimulation, Bone
(7.O8) THE EFFECT OF ALTERING FREQUENCY
DISTRIBUTION OF MECHANICAL STIMULATION ON
MYOCARDIAL-EQUIVALENT TWITCH FORCE
Ye KY (1), Black LD III (1)
1. Tufts University
Tissue engineering of myocardium represents a promising
approach for the treatment of myocardial infarcts.
Previously, we have shown that cell-induced alignment
improved cellular communication via increased Cx43
functionality, resulting in an increase in twitch force
beyond that of merely aligning the cells. Previous work by
other groups in the field has also shown that periodic
mechanical stimulation improves the observed twitch
force; however, these studies were carried out using
constant frequency and amplitude. It has been shown in
other tissues that variations in the stretch amplitude can
improve matrix deposition and protein synthesis. The
induced stretch in myocardium mimics the stretching of
the ventricle as it fills with blood. Since blood pressure
and heart rate follow a Gaussian distribution, we
hypothesize that normally varying the stimulation
frequency would improve twitch force compared to
uniformly distributed and constant frequency stimulation.
To test this, neonatal rat cardiac cells were entrapped in a
tubular fibrin gel and cultured in a custom distension
bioreactor for 14 days. A computer program controlled
the stimulation frequencies according to user-chosen
distributions during cell culturing. The constructs were
mechanically stimulated with constant frequency (C),
Gaussian frequency distribution (G), and uniform random
frequency distribution (R). Static culture (S) was used as a
control. A preliminary analysis of twitch force data found
that C (4.4±1.6mN) constructs improved twitch force over
G (1.6±1.1mN), R (2.7±1.1mN), and S (1.1±1.1mN)
constructs with statistical significance (P<0.05). Ongoing
experiments are being conducted to determine whether
this improved function is the result of enhanced cell
viability, improved cell communication or increased
contraction efficiency. Future experiments include
varying the amplitude in accordance with changes in the
frequency to evaluate further differences in twitch force,
as well as investigating whether mechanical stimulation
enhances the function of engineered myocardium
created with MSC derived cardiomyocytes.
Keywords. Cyclic Distension, Engineered Heart Tissue,
Variable Stretch, Fibrin Gel
(7.O9) DEVELOPMENT OF AN NOVEL BIDIRECTIONAL
CONTINUOUS PERFUSION BIOREACTOR (BCFB), FOR
CULTURING CELLS IN 3D SCAFFOLDS
Gardel LS (1,2), Dias A (1), Link D (1), Serra LA (3), Gomes
ME (1), Rui RL (1)
1. 3B's Research Group, IBB; 2. ICBAS-UP; 3. Departament
of Ortophysiatric, General Hospital Santo António, Porto,
Portugal
This works presents a new bioreactor, for the culturing of
3D scaffolds aimed at applications in bone tissue
engineering. The Bidirectional Continuous Perfusion
Bioreactor (BCFB) promotes the mechanical stimulation
of cells through the creation of shear forces induced by
flow perfusion, using different pressure gradients,
controlled by a peristaltic pump. Additionally it provides
the possibility of varying both perfusion flow rate/flow
direction. The main innovation consists in the possibility
of culturing scaffolds of large dimensions, as the control
of flow perfusion and pressure gradient in the
inside/outside of the scaffold, enables a culture
environment that favours the access to nutrients and
removal of metabolic wastes of the cells located in the
inner regions. Starch/Polycaprolactone (SPCL) fibbers
mesh scaffolds (14 samples with 16mm x 4mm thickness
with a concentric hole of 6mm) were seeded with 1x106
goat marrow stromal cells and stacked, completing a 48
mm thick construct. After 14 and 21 days of culture in the
bioreactor at a flow rate of 1 ml/min, the samples were
collected for DNA/ALP concentration, and SEM. Static
cultured constructs were used as controls.
The results showed higher ALP activity levels in dynamic
cultures than those obtained under static conditions.
However, the number of cells (obtained from DNA
amounts) in constructs cultured in the bioreactor showed
lower values compared to static cultures, showing that
static conditions tend to privilege the metabolic way for
cellular proliferation while dynamic conditions tend to
privilege
the metabolic way
for osteogenic
differentiation. The lower values of the DNA amount of
the constructs in the bioreactor could be explained by
shear forces in the constructs, thereby hampering cell
proliferation but enhancing cell differentiation. The BCFB
can be used for enhancing cellular differentiation and
proliferation by applying flow perfusion. Therefore, this
bioreactor could be applicable to generate large-sized 3D
scaffolds.
Keywords. Bioreactors; Bone Tissue Engineering; 3D
scaffolds Large Dimensions
expanded in DMEM with 1%antibiotics-10%fetal calf
serum (DMEM+) and characterised by differentiating
them down the adipogenic and osteogenic lineages.
Seeding studies were conducted for both scaffolds.
Seeded scaffolds were either statically cultured in well
plates or in the PBRS with a flow rate of 0.75mL/min,
both with DMEM+. At days 4, 7 and 14 cell proliferation
(AlamarBlue and DNA assays, n=3), osteogenic
differentiation (ALP assay, n=3) and cell distribution
(histology) were analysed. Constructs were visualised by
SEM.
Results. Statistically significant increased cell proliferation
(p≤0.05) was seen in samples cultured under flow
perfusion conditions for both scaffolds at all times. ALP
activity was significantly higher (p≤0.05) in the bioreactor
constructs at all times points for both scaffolds.
Histological analysis revealed a more even cellular
distribution in the constructs cultured in the PBRS. The
development of a cell layer over time was observed by
SEM.
Conclusions. The PBRS used in this study increases cell
proliferation and osteogenic differentiation and improves
cell distribution throughout the scaffolds. We conclude
that the development of constructs for bone tissueengineering purposes can be achieved by using a PBRS.
Keywords. flow perfusion bone
SEM photo at day 4 of flow perfusion culture of CaP-Ti
cylinder where cells have proliferated.
(7.O10) A PERFUSION BIOREACTOR SYSTEM FOR THE
DEVELOPMENT
OF
TISSUE-ENGINEERED
BONE
CONSTRUCTS
García E (1), Hua J (1), Rayan F (1), Blunn G (1)
1. University College London (UCL), UK
Introduction. The development of tissue engineered
bone constructs is of considerable importance to fill
defects associated with segmental bone replacement in
bone cancer or spinal fusions.
Aim. To culture mesenchymal stem cells (MSCs) on
porous and granulated scaffolds using a perfusion
bioreactor system (PBRS) and study their proliferation,
osteogenic differentiation and distribution compared to
statically cultured constructs.
Hypothesis. A PBRS will provide an even distribution of
MSCs throughout porous and granulated scaffolds and
will enhance MSCs proliferation and osteogenic
differentiation compared to statically cultured scaffolds.
Methods. An easily sterilised and assembled PBRS was
designed and implemented. The scaffolds were Silicon
substituted hydroxyapatite granules (Si-HA) and calciumphosphate coated Ti6Al4V porous cylinders (CaP-Ti).
Ovine MSCs were isolated from bone marrow aspirates,
(7.O11) THE IMPORTANCE OF GRADIENTS IN ARTICULAR
CARTILAGE
Spitters TWGM (1), Fernandes H (1), Liu J (1), van
Blitterswijk CA (1), Karperien M (1)
1. Department of Tissue Regeneration, MIRA Institute,
University of Twente, The Netherlands
It is hypothesized that gradients of growth factors (GFs)
and GF antagonists exist in articular cartilage and play an
important role in the balance between anabolic and
catabolic processes. It is believed that such gradients are,
at least partially, responsible for the zonal organization of
articular cartilage. Despite their importance, current
bioreactor designs for articular cartilage tissue
engineering have limited options for introducing GF and
GF-antagonist gradients. To address this issue we have
developed a dual flow bioreactor which can
accommodate
four
articular
cartilage
cubes
(4.5x4.5x3mm) between two medium compartments. The
reactor was designed in such a way that it mimics the
knee joint as good as possible. The top and bottom
compartment are mimicking the synovial fluid and
subchondral bone respectively. The bioreactor was
complemented with a plunger that was attached to a
compression insert. In this way load can be applied from a
vertical position (Figure 1A).
Computational fluid dynamics was used to predict the
occurrence of an oxygen gradient, which is shown in
figure 1B. The model was then evaluated with a cell line
containing a reporter system consisting of a HRE element
controlling GFP expression. Medium in the top and
bottom compartment were saturated with a different
oxygen concentration. Quantification of the GFP
expression showed the occurrence of an oxygen gradient
(Figure 1C+D).
In conclusion, this unique bioreactor design assists in
creating gradients, as shown for oxygen, and it will be
used for creating gradients of growth factors and
regulatory molecules. The ability to manipulate these
gradients can aid in creating an ex vivo environment
which may support the engineering of the native
structure of articular cartilage.
Keywords. bioreactor, gradient, oxygen
(7.O12) NUMERICAL ANALYSIS OF NUTRIENTS
TRANSPORT IN CONVECTION-ENHANCED HFMBS FOR
LONG BONE TISSUE ENGINEERING
Zanetti EM (1), De Napoli IE (1), Audenino AL (2),
Catapano G (1)
1. Università della Calabria; 2. Politecnico di Torino
Introduction. Recent experimental evidence shows that
delocalized and distributed nutrients supply and high
spontaneous Starling flows in hollow fibre membrane
bioreactors (HFMBs) yield cm-scale BMSC aggregates,
possibly by relieving nutrients limitations typical of other
bioreactors for bone tissue engineering (BTE). The
difficult non-intrusive measurement of nutrients and cell
concentrations during culture makes mathematical
modelling of mass transport, cell growth and metabolic
reaction kinetics very attractive: to analyze the effects on
cell organization and growth of nutrients transport, cell
seeding and bioreactor geometry and operation; and to
optimize bioreactor design and operation. Unfortunately,
the non-uniform cell distribution observed in culture
experiments and high Starling flows render most
proposed models inadequate to the purpose. This paper
presents mathematical models of HFMBs operated in
close shell mode covering the range from diffusionlimited to convection-dominant nutrients transport
conditions for both uniform cell distribution and the
actual non-uniform cell distribution observed in
experiments with BMSCs at different culture times.
Methods. Models are based on a multi-compartment
description of HFMBs based on the Krogh cylinder
assumption, and on a quasi-steady state analysis of
evolution of nutrients and cell concentration profiles.
Relevant non-dimensional parameters were identified,
and governing momentum and mass transport equations
were numerically solved with a finite element commercial
code with particular reference to oxygen and glucose.
Where possible, parameters assessed from culture
experiments were used.
Results and conclusions. Simulation results demonstrate
the importance of convective nutrient transport,
membrane permeability and packing density in the cell
compartment. They also suggest that bioreactor
operation should be changed during culture to adapt to
the variable nutrients demand of cells in the HFMB shell,
as they proliferate and aggregate in 3D structures slowly
filling up the shell space and exhibiting a Darcy
permeability increasing in time.
Keywords. Nutrient transport; Bone tissue; Hollow fibre
membrane bioreactor
(7.O13) VESSEL METABOLISM UNDER MECHANICAL
LOAD - IMPLICATIONS FOR VASCULAR TISSUE
ENGINEERING
Hoenicka M (1), Schrammel S (2), Puehler T (1), Hirt S (1),
Birnbaum DE (1), Schmid C (1)
1. Department of Cardiothoracic Surgery, University of
Regensburg Medical Center, Regensburg, Germany; 2. FB
Maschinenbau,
University
of
Applied
Sciences
Regensburg, Regensburg, Germany
Introduction. Tissue engineered prostheses like vascular
grafts or heart valves are usually generated in perfusion
bioreactors which provide mechanical stimuli to condition
the constructs. To assess whether conditioning alters
nutritional requirements, we investigated the effects of
shear forces and luminal pressure in a vessel model.
Methods. Bovine saphenous veins were perfused in mock
circulations for 4 days. Group 1 vessels were perfused
with M199 at 40ml/min. Group 2 vessels were subjected
to increased shear forces (+12% dextran). Group 3 vessels
were additionally challenged by increased luminal
pressure (+20mm Hg). The corresponding groups 1', 2',
and 3' were endothelium-denuded before perfusion.
Substrate conversion was calculated from glucose and
lactate levels. Blood gases were measured upstream and
downstream of the samples. Contractile function and
tetrazolium dye reduction were determined before and
after perfusion.
Results. Noradrenaline-induced contractions after
perfusion were significantly stronger in group 3 vessels
and significantly lower in denuded vessels. Tetrazolium
dye reduction was attenuated in groups 1'-3'. Glucose
was converted stoichiometrically to lactate except groups
3, 1', and 3' which produced more lactate than glucose
could supply. Oxygen concentrations were unaltered
between vessel inlet and outlet except in group 2.
Conclusions.
Vessels
did
not
use
oxidative
phosphorylation but lactate fermentation to meet their
energy needs. Luminal pressure but not increased shear
forces alone improved contractile function after perfusion
and induced the consumption of substrates other than
glucose in an endothelium-independent fashion.
Conditioning bioreactors may thus deplete perfusion
media of substrates more rapidly and in different
patterns compared to static cultures, and may in fact call
for media tailored for this purpose, whereas oxygen
partial pressures can be adjusted freely to support tissue
growth optimally.
Acknowledgements. This study was funded by Deutsche
Forschungsgemeinschaft (BI 139/2-1, HA 4380/5-1, and LI
256/68-1).
Keywords. bioreactors; metabolism; pressure; shear
forces
(7.O14) DYNAMIC EXPANSION OF HUMAN UMBILICAL
CORD CELLS IN A ROTATING BED SYSTEM BIOREACTOR
FOR TISSUE ENGINEERING OF HUMAN HEART VALVES
Reichardt A (1), Hetzer R (2), Lüders C (1)
1. Department of Cardiothoracic and Vascular Surgery
and Laboratory for Tissue Engineering, Deutsches
Herzzentrum Berlin, Augustenburger Platz 1, Berlin; 2.
Deutsches Herzzentrum Berlin, Augustenburger Platz 1,
Berlin
Introduction. To overcome limitations in static cell
culture systems the dynamic expansion of cells could be
an important tool for the tissue engineering of human
heart valves. Dynamic expansion should provide
continuous perfusion of the cells, large numbers of viable
pre-conditioned cells after a short time period and
controllable environmental conditions; it should also be a
reproducible process. For this purpose human umbilical
cord myofibroblasts were cultivated and expanded in a
rotating bed system bioreactor.
Methods. Myofibroblasts isolated from human umbilical
cord arteries (12x106cells) were cultured for 9 days under
hypoxic conditions in a bioreactor system which consists
of a cylindrical culture vessel with an integrated rotating
bed of several polycarbonate slides. Via an integrated
control unit several parameters were measured
throughout the fabrication process to achieve optimal
culture conditions. Perfusion and slow bed rotation
minimized mass transfer limitations and therefore
supported the cells with sufficient nutrients. Feeding
leads to continuous medium exchange in the culture
vessel. Tapping for medium samples allowed the amount
of nutrients and metabolic waste products i.e. lactate to
be controlled. The cells were characterized by a specific
surface marker profile using flow cytometric analysis
before and after cultivation in the bioreactor system.
Results. Myofibroblasts were successfully expanded by
the factor 30. The fast cell growth possessed a large
number of viable cells for tissue engineering applications.
There was no change in the expression of cell surface
markers after cultivation in the bioreactor compared to
the expression before.
Conclusion. Expansion of large numbers of viable cells
was realized in an easily manageable and controllable
bioreactor system in a short period of time, with
minimized effort and labor costs. In future applications
the dynamic expansion of cells will be an important tool
for the tissue engineering of human heart valves.
Keywords. Tissue Engineering, Bioreactors, Dynamic
cultivation of cells
(7.O15) A NOVEL CONTROL UNIT TO CULTURE
MESENCHYMAL STEM CELLS UNDER CONTROLLED AND
REPRODUCIBLE CONDITIONS IN A PERFUSION
BIOREACTOR
Kress S (1), Lavrentieva A (1), Martin y (2), Tappe A (2),
Scheper T (1), Kasper C (1)
1. Leibniz University of Hanover, Institute of Technical
Chemnistry; 2. Sartorius AG
Bioreactors are required in Tissue Engineering to ensure
controlled and stable conditions for the fabrication of
engraftable tissues. This includes the monitoring and
regulation of the temperature, pH and pO2, as well as
mass transportation of nutrients and waste material.
Moreover a bioreactor should mimic the natural
environment as accurately as possible. In addition
mechanical stimulation during the cultivation performed
by a special bioreactor can support the proliferation and
differentiation of human mesenchymal stem cells.
Therefore we developed a control unit to guarantee
reproducible conditions for bioreactor cultivations in the
area of Tissue Engineering. The system consists of a
control tower with a GMP conform software, a stirred
tank bioreactor (STR), a perfusion bioreactor and a
heating cabin. A perfusion bioreactor with 3Dbiomaterials has been used for cell culturing to imitate
the fluid shear stress in bone tissue. Moreover perfusion
bioreactors reduce the limitation of mass transportation,
because the media is continuously transported through
the 3D-biomaterials. The culture media is preconditioned
in the STR due to a combination of air, nitrogen and
carbon dioxide prior pumping it through the perfusion
bioreactor. Thus the pH and the pO2 value can be
adjusted. The temperature of the culture media is
regulated by a heating mat below the STR; moreover the
perfusion bioreactor is setup in a heating cabin. The
glucose and lactate values can be measured offline and if
required fresh media can be added into the STR and
waste media be removed.
Human mesenchymal stem cells have been cultivated for
3 weeks on 3D-biomaterials in a perfusion bioreactor
whereat the media conditions where adjusted by the
control unit. The proliferation of the cells has been
demonstrated by the consumption of glucose and by the
MTT activity test. The pH and the pO2 values have been
recorded by the GMP conform software.
Keywords. mesenchymal stem cells, bioreactors,
perfusion, control unit
(7.P1) EFFECT OF PERFUSION CULTURE SYSTEM ON IN
VITRO OSTEOGENESIS OF HUMAN MESENCHYMAL STEM
CELLS SEEDED ON POROUS HYDROXYAPATITE
Saino E (1), Bloise N (1), Spinelli L (2), Mantero S (2),
Martinetti R (3), Imbriani M (4), Visai L (1)
1. Department of Biochemistry, University of Pavia, Italy;
2. SKE Advanced Therapies S.r.l., Milano, Italy; 3. FinCeramica Faenza S.p.A., Faenza, Italy; 4. Salvatore
Maugeri Foundation IRCCS, Pavia, Italy
Introduction. Dynamic culture yields an excellent
homogeneous distribution of cells and matrix, and shear
stresses applied by medium stimulate the cells to
proliferate and differentiate, ensuring continuous
nutrition of cells and removal of waste products (1). The
aims of the study were to test the proliferation and
differentiation of human Mesenchymal Stem Cells
(hMSCs) cultured on porous hydroxyapatite (HA) scaffolds
and to compare conventional static culture to dynamic
flow perfusion culture.
Materials and Methods. Porous hydroxyapatite (HA)
scaffolds were provided by Finceramica Biomedical
Solutions [ENGI (SLV002005) ST] (cylindrical form
Ø=10mm and H=4 mm with an inner porosity close to
80±5 vol.%). HMSCs were isolated from BM as previously
described (2) and seeded on HA scaffolds. The perfusion
bioreactor was (Fig. 1) designed and developed by SKE
Advanced Therapies S.r.l. The cells/hydroxyapatite
construct was perfused for 21 days in osteogenic
medium. The flow was monodirectional (100µm/sec). pH
culture medium was misured using an optical sensor
(Fluorometrix,MA,USA). Cell viability was determined by
MTT assay. Calcium content, alkaline phoshatase (ALP)
activity and bone extracellular matrix proteins were
evaluated as described (3).
Results. MTT assay showed an increase of the living cells
in the perfused culture. In agreement with this results, an
enhancement of ALP activity, mineralization and bone
proteins deposition were observed in the perfused
culture.
Conclusions. These results demonstrate the feasibility
and benefit of culturing cell/HA constructs in a flow
perfusion bioreactor for bone tissue engineering
applications.
Acknowledgements. This work was supported by "Project
SAL-45" financed by Regione Lombardia and by project
financed by FONDAZIONE ALMA MATER TICINENSIS
(2010).
References.
(1) Cartmell SH et al. Tissue Eng. 9: 1197-1203, 2003.
(2) Bernardo ME et al. J Cell Physiol, 211: 121-130, 2007.
(3) Saino E et al. Eur Cell Mater. Jan 14; 21: 59-72, 2011.
Keywords. Perfusion bioreactor, Human Mesenchymal
Stem Cells, Porous hydroxyapatite scaffolds, osteogenic
differentiation
(7.P2)
THREE-DIMENSIONAL
CULTIVATION
OF
OSTEOBLASTS IN LARGE SCAFFOLD USING RADIAL-FLOW
BIOREACTOR
Yoshinari M (1), Arano T (1), Igarashi T (1), Matsuzaka K
(1), Inoue T (1)
1. Tokyo Dental Collage
Introduction. Bioreactors employing different types of in
vitro physiological cell stimulation have been developed
to obtain three-dimensional cultivation for tissue
engineering. The purpose of this study was to determine
whether osteoblastic cells proliferated uniformly over a
large scaffold with a diameter of 18 mm and height of 10
mm under dynamic cultivation with the radial-flow
bioreactor (RFB), and thereby ascertain the potential of
this system in the regeneration of jaw bone.
Methods. Mouse osteoblastic cells (MC3T3-E1) were
seeded onto type-1 collagen sheets. Cells were then
incubated outside the reactor for 6 hours to produce precultured sheets. The 6 pre-cultured sheets were then
placed in the RFB to fabricate the large scaffolds. Cells
were dynamically cultured for one week at 37
˚C, pH 7.4,
DO 6.86 ppm, and with the culture medium circulating at
3 mL/min. For static cultivation, cells were cultured in the
same manner without circulating culture medium. The
resulting cell proliferation and cell distribution were
analyzed.
Results. After 6 hours of pre-culturing, most of cells were
remained in the collagen sheets, and 97% of the cells
were still alive and capable of proliferation. This suggests
that the pre-culturing system is an effective method for
providing viable cells for further dynamic culture. After
one week of dynamic cultivation, osteoblastic cells
showed uniform proliferation with yielding a large
number of cells more than 5 times greater than that
obtained with static cultivation.
Conclusions. These results indicate that the RFB is a
promising system for three-dimensional cultivation of
osteoblastic cells for treating large bone defects by tissue
engineering.
Acknowledgements. This research was supported by Oral
Health Science Center Grant HRC7 from Tokyo Dental
College, and by a “High-Tech Research Center” Project for
Private Universities: matching fund subsidy from MEXT of
Japan, 2006-2011.
Keywords. radial-flow bioreactor, osteoblasts, large
scaffold
(7.P3) A NEW STRETCHING BIOREACTOR FOR DYNAMIC
ENGINEERING OF MUSCLE TISSUES
Giraud MN (1), Fouassie r C(1), Guex G (1,2), Näther S (3),
Fortunato G (2), Carrel TP (1), Tevaearai HT (1)
1. Inselspital; 2. Empa; 3. University of Bern.
Objectives. We aim to define in vitro dynamic culture
conditions to improve cell density and organisation of
engineered muscle construct. We report here our ongoing study on the development and validation of a new
device for the generation of stretch culture conditions.
Methods. Custom made silicon bulb (produced with a
water-soluble wax mold) were covered with electrospun
poly-caprolactone (PCL) micron-scaled fiber matrix. Pump
controlled volumetric changes induced bulb enlargement
and resulted in matrix stretching. Spatial characterisation
of the stretch was analysed using a GOM 3D digitizer and
GOM ARAMIS software.. C2C12 cells were seeded on the
matrix and cultured for 1 week under mechanical
stimulation. Static, cyclic (1Hz) and ramp (cycles of 24h
stretch /24h rest) stretch with strain conditions were
applied. Cellular responses were investigated by scanning
electronic microscopy, immunostaining and 3D confocal
analysis.
Results. 1: A gradient of surface strains was characterised
from the base to the apex of the bulb. When inflated, the
apex showed a linear increase in the strain from 4 to 23%.
Meanwhile, the increase of strain at the base was limited
and ranged from 2% to 8%.
2: Compared to static culture conditions, dynamic culture
induced cellular multilayer formation. This effect
appeared to be dependant of the applied stretch
amplitude. Ramp stretch with low strain (gradient from 3
to 5%) induced a 2-time thickening of the tissue compare
to higher stretch (gradient strain from 6 to 12%). Ramp
stretching is associated with randomly oriented cells. In
opposite, cyclic strains improved cell orientation.
Conclusion. We provide preliminary evidence that our
new device composed of a bulb shape carrier covered
with microfibers matrices is promising for structured
muscle tissue generation. In addition, stem differentiation
and in vitro modelisation of cardiac remodelling are other
possible fields of investigation that may benefit from our
device.
Keywords. muscle biografts; bulb carrier; electrospinning
(7.P4) HYDROSTATIC PRESSURE IMPROVES BONE CELL
MORPHOLOGY AND GENOTYPIC EXPRESSION IN
DYNAMIC CULTURE
Merzari E (1), Carletti E (1), Floren ML (1), Maniglio D (1),
Motta A (1), Migliaresi C (1)
1. University of Trento, Department of Materials
Engineering and Industrial Technologies and Biotech
Research Center
The ability to control and influence cell behavior to
produce functional tissues is critical in tissue engineering
and regenerative medicine. Mirroring both the natural
structure and morphology of the native tissue as well as
imitating the complex events of the cellular microenvironment is vital for the success of an engineered
tissue. Beyond enhancing nutrient diffusion and cell
growth, bioreactors are often employed to administer
mechanical stimuli to cell cultures with the aim to mimic
the stresses observed naturally in vivo. In particular, bone
tissue is remarkable in that it has the capacity to adapt its
form, i.e. density and internal architecture, in response to
mechanical stimulation. Consequently, it has been shown
that the application of various dynamic stresses, such as
shear and strain, can influence both bone cell genotype
and ECM production; however, these complex events
linking mechanotransduction to cellular activity are still
elusive. To elucidate this phenomena we developed a
dynamic culturing method that utilizes hydrostatic
compression to stimulate cell substrates. By controlling
the frequency, magnitude and even cycle of the applied
stress, we aimed to investigate the response of such
stimuli on the proliferation, migration and genotypic
expression of bone cells.
The poly(D,L-lactic acid) (PDLLA) porous scaffolds utilized
were prepared using a salt-leaching method in which
scaffolds were tailored to meet specific porosity and pore
size requirements. Biological evaluation was carried out
using Alamar blue assay for proliferation and visual
inspection by confocal laser microscopy (CLSM). RT-PCR
was employed to map the cell gene expression during
dynamic conditions providing information on matrix
production and mineralization, both of which are critically
important in the formation of bone tissue.
Preliminary results indicate that cellular activity is
enhanced in dynamic culture compared to static controls.
This is observed via increased bone genotypic expression
as well as bone matrix protein production.
Keywords. Bioreactor, bone tissue engineering, genotypic
expression
(7.P5) A USER-FRIENDLY MULTI-CHAMBER PERFUSION
PLATFORM: PRELIMINARY TESTS WITH THREEDIMENSIONAL POROUS PCL SCAFFOLDS
Piola M (1), Cantini M (2), Sadr N (1), Gómez Ribelles
JL (2), Ferrario G (3), Soncini M (1), Fiore GB (1)
1. Politecnico di Milano, Dipartimento di Bioingegneria,
Milano, Italy; 2. Centro de Biomateriales e Ingeniería
Tisular, Universidad Politécnica de Valencia, Valencia,
Spain; 3. Università di Milano, Dipartimento di Scienze
Cliniche L. Sacco, Milano, Italy
Introduction. Several flow perfusion bioreactors have
been documented for dynamic cell culture within threedimensional (3D) matrices [1]. Flow perfusion ensures
adequate nutrient supply/waste removal within the
substrate, and suitable stimuli to the cells, representing
an appealing tool to replicate natural tissue
microenvironments. In this work, we develop a userfriendly, GMP-compatible, multi-chamber, confined-flow
perfusion platform in close collaboration with physicians
and biologists, providing a simple and straightforward
tool for dynamic cell cultures. Bioreactor testing was
carried out using line and primary cells.
Methods. The device (Fig.1A) consists of a six-chamber,
stand-alone platform able to manage several
independent and simultaneous experiments in controlled
culture conditions. Each culture chamber (Fig.1B) consists
of a housing, a silicone cartridge that, by virtue of its
deformability, acts as a watertight scaffold holder, and a
7-ml medium reservoir coupled with a disposable vented
screw cap. The device compact size, the extremely small
number of components and the use of bayonet couplings
allow a simple, fast, and sterile assembly by the operator.
In order to investigate the bioreactor performances, oneway and oscillatory seeding experiments are performed
on porous ε-polycaprolactone scaffolds with MC3T3-E1
cells and primary human fibrocytes. Cell adhesion and
distribution within the scaffold are adopted as bioreactor
performance read-out.
Results. Experimental campaigns with 3D matrices allow
us to determine that: i) seeding perfusion rate in the
range 0.03-0.1 ml/min improves cell seeding efficacy
compared to static seeding, and ii) both one-way (Fig.1C)
and oscillatory cell seeding (Fig.1D) result in a uniform
distribution of cells within the scaffold.
Conclusions. The developed bioreactor is functional,
versatile, and straightforward. The preliminary in vitro
tests prove the efficacy of the system in enhancing cell
seeding efficiency, opening the way for further studies
addressing long term colonization of the scaffold.
Reference. [1] Martin I et al., Trends in Biotechnology; 22
(2004): 80-86
Keywords. Confined low perfusion bioreactor; multichamber platform; three-dimensional scaffold; dynamic
cells seeding
constructs’ secretome is needed upon implantation to
defect site as the pre-culture period could influence the
construct’s integration into the host.
Keywords. Stem cells; Bioreactor; Growth factors;
Cytokines
(7.P6) TIME-COURSE EXPRESSION OF VEGF, FGF-2, AND
IL-11 BY HUMAN MESENCHYMAL STEM CELLS UNDER 3D
CULTURE IN FLOW PERFUSION BIOREACTOR
Sladkova M (1), Vandamme K (1,2), David B (3), Petite H
(1)
1. Univ Paris Diderot, Sorbonne Paris Cité, Faculty of
Medicine, Laboratory of Bioengineering and Biomechanics
for Bone Articulation (B2OA), UMR CNRS 7052, F-75010
Paris,
France;
2.
BIOMAT
Research
Cluster
K.U.Leuven/Department of Prosthetic Dentistry, Faculty of
Medicine, Kapucijnenvoer 7 Blok a bus 7001, 3000 Leuven,
Belgium; 3. École Centrale Paris, Mechanics, Structures
and Materials (MSSMat), UMR CNRS 8579, F-92295
Chatenay-Malabry, France
The beneficial effects of delivered mesenchymal stem
cells (MSCs) to defect site are related not only to their
multipotency but as well to their trophic action. We
hypothesized that the release of signaling molecules
could be modulated by culture conditions. The objective
of this study was to evaluate the time-course expression
of angiogenic growth factors vascular endothelial growth
factor (VEGF) and fibroblast growth factor 2 (FGF-2) and
anti-inflammatory cytokine interleukin-11 (IL-11) by
human MSCs under three-dimensional (3D) culture in
flow perfusion bioreactor.
Human 105 MSCs (hMSCs) were seeded per coral cube
(3x3 mm). The coral-containing hMSCs (“constructs”)
were cultured in custom-made flow perfusion bioreactor
either under static (control) or dynamic flow perfusion
culture conditions. At day 0, 7, 14, and 21 the collected
constructs and medium underwent analysis to assess
gene and protein expression, respectively. Under dynamic
conditions VEGF protein level was significantly lower
(p<0.01); however, the gene expression was significantly
lower after 7 days (p<0.034) and higher after 14 and 21
days (p<0.0039; p<0.049, respectively) compared to static
condition. FGF-2 was not detected at protein level;
however, its gene expression was significantly lower after
7 days (p<0.00021) under dynamic over static condition.
The IL-11 protein level was significantly increased after 14
and 21 days (p<0.01); however, its gene expression was
significantly lower after 7 days (p<0.00067) and higher
after 14 days (p<0.011) under dynamic over static
condition.
To the best of our knowledge, the present study provides
the first evidence about the time-course expression of
VEGF, FGF-2, and IL-11 by hMSCs in 3D flow perfusion
culture. Moreover, it shows that gene and protein level of
studied molecules significantly depend on culture
condition applied. A deeper characterization of hMSCs
(7.P7) A MULTI-LAYER MICROFLUIDIC CHAMBER TO
CULTURE UNIFORM-SIZEDCHONDROCYTE PELLETS
Piraino F (1), Pierro M (1), Moretti M (2,3), Redaelli A (1),
Rasponi M (1)
1. Bioengineering Department, Politecnico di Milano,
Milano, Italy; 2. Gruppo Ospedaliero San Donato
Foundation, Milano, Italy; 3. IRCCS Istituto Ortopedico
Galeazzi, Milano, Italy
Introduction: In the last decades the biological field has
spent a steady effort in reducing experimental systems
from a macro- to a micro- or lab-on-a-chip scale [Bauer M
et al., IntegrBiol 2010]. Only recently, researchers started
turning to 3D cell cultures, which better reproduce the in
vivo-like cell microenvironment [Abbott A, Nature 2003].
However, generation and culture of cell aggregates at the
microscale still remainschallenging.Main goal of this work
was the generation of uniform-sized 3D chondrocyte
pellets within a microfluidic-perfused environment.
Methods: A PDMS microfluidic device was developed,
consisting of two layers: a top layer containing a straight
channel 40mm long provided with two chambers, used
for bubble trapping purposes, and a bottom layer,
containing a microfluidic chamber for cell collection and
pellet formation.After a culture period of 14 days, pellets
were analyzed for cell metabolic activity, sGAG and DNA
content and the ECM formation was assessed through
histology. Control pellets were also obtained and cultured
with standard protocols.
Results: Main advantage of the chamber design was the
ability to induce pellet generation by means of gravity
sedimentation without the need of centrifugation
7
steps.Cells, injected at a concentration of 2x10 cells/ml,
filled the chamber (Fig.1A) and, after 20 hours,
aggregated in uniform-sized pellets (Fig.1F). Pellets
obtained with the microfluidic system showed a
comparable viability to the controls. Moreover,
microfluidic pellets were homogeneously populated with
spaced cells with interposed matrix.
Conclusions: The methodology described in this work is
simple and may be scaled up for culturing large numbers
of pellets in a single device. Although this study focused
on chondrocytes, the technology described is versatile
and should be readily applicable to other cell types in a
physiologic-like 3D setting.
Acknowledgments: This project partly was supported by
Cariplo Foundation and ProgettoRocca.
Keywords. Microfluidics, Chondrocyte, Pellets, Perfusion
8. CARTILAGE
Chair: Pedro Guillén-García
Co-chair: Pedro Hernández-Cortés
Keynote speaker: Pedro Guillén-García
Organizer: BioIbérica
Synopsis: Autologous chondrocyte implantation is a wellestablished method for the treatment of several chondral
defects, although the long-term clinical results of this
type of therapy are controversial, and several researchers
previously demonstrated that the clinical results of cell
therapy using cultured chondrocytes are highly variable in
relationship to several factors.
In this symposium, several topics related to the novel
therapies applied to the regeneration of the human
cartilage will be discussed. Some of the most relevant
topics of this symposium are:
− Chondrocytes culture conditions
− Chondrocytes markers
− Bioreactors for chondrocyte culture
− Extracellular matrix in cartilage
− Biomaterials
− Clinical chondrocyte implants (ACI)
− Clinical chondrocytes implants using membranes
(MACI)
− Clinical trials in cartilage repair
We expect that all scientists, clinicians and professionals
involved in the field of cartilage biology, are invited to
submit abstracts to this symposium.
(8.KP)
THE
TREATMENT
WITH
AUTOLOGOUS
CHONDROCYTES IS NOWADAYS THE ONLY TECHNIQUE
THAT REPLICATES THE NORMAL CARTILAGE AFTER A
LESION
Guillén-García P (1)
1. Clinica CEMTRO, Madrid, Spain
Introduction. Articular cartilage damaged has limited
potential to heal and if defects are left untreated, they
may progress to osteoarthritis. In past decades, research
was focused in developing techniques for stimulating
cartilage repair and regeneration, in particular cell
therapy techniques as autologous chondrocyte
implantation (ACI). Another approach is the use of in vitro
engineered tissue obtained using cells seeded onto a
biocompatible membrane. This procedure is called MACI
(Matrix-Induced Autologous Chondrocyte Implantation)
and can be combined with arthroscopy. We describe our
8-year experience with MACI, presenting follow-up data
from 50 patients.
Material and Methods. We present the results obtained
in 150 consecutive patients, evaluated by an in-house
validated clinical protocol which included a survey stating
the following data: age, sex, location and of the defect,
affected limb, number and type of previous surgeries,
mobility after MACI implantation and time of sick leave.
In 50 cases, a second biopsy was performed in a mean
follow-up period of 2 years.
Results. In 126 patients the lesion was located in the knee
and in 24 in the ankle. Arthroscopic MACI was carried out
in 53 of them while in the remaining 97 an open-fashion
procedure was followed. The histological study of the
novel tissue formed revealed an architecture of hyalinelike cartilage in all patients, although the number of cells
was lower than the normal hyaline cartilage. All the
biopsies analyzed expressed the aggrecan, COL I and COL
II genes.
Conclusion. The implantation of autologous chondrocytes
is a good procedure to treat chondral and osteochondral
lesions in the knee and ankle, preserving the integrity of
the joints.
Keywords. ACI, MACI, autologous chondrocyte
implantation, second look
(8.O1) ANISOTROPIC FIBROUS TISSUE SCAFFOLDS FOR
ARTICULAR CARTILAGE REGENERATION
McCullen S (1), Autefage H (1), Callanan A (1), Stevens M
(1)
1. Imperial College London
Introduction. Articular cartilage is a highly organized,
fibre-reinforced tissue with a complex extracellular matrix
of proteoglycan molecules retained within a fibrillar type
II collagen meshwork. The structural arrangement of the
collagen fibre network provides the tensile reinforcing
elements of cartilage and exhibits unique anisotropic
(depth-dependent) organization. The superficial, middle
and deep zones of cartilage feature varying collagen II
alignment as well as decreasing, depth-dependent tensile
properties. Current cartilage tissue engineering solutions
fail to mimic this zonal organization; thus the goal of this
work was to fabricate anisotropic electrospun constructs
that mimic the native fibre organization and tensile
properties of articular cartilage.
Methods. Anisotropic electrospun scaffolds were
fabricated by electrospinning poly(ε-caprolactone) (PCL)
while gradually varying the polymer concentrations (15 or
25 w/v%) and the speed of a rotating mandrel (2000 rpm
vs. 100 rpm) to collect either aligned or random fibre
networks, respectively. The resulting layered constructs
were assessed via electron microscopy, tensile testing,
and their ability to support in vitro chondrogenesis of
bovine chondrocytes.
Results. Anisotropic constructs were created by
sequentially electrospinning different PCL solutions. 3D
constructs were generated, featuring variations in fibre
morphology, orientation, and tensile properties,
mimicking the morphology and mechanical behaviour of
articular cartilage (Figure 1). Zonal tensile strength of the
anisotropic construct decreased within each layer as
indicated in Figure 1: zone B (35 MPa), zone C (7.4 MPa),
and zone D (5.7 MPa). Bovine chondrocytes were able to
adhere, proliferate and differentiate on the scaffolds for 5
weeks in vitro on both homogenous and anisotropic
constructs with depth-dependent tensile properties (data
not shown).
Conclusions. We have fabricated the first anisotropic
fibrous construct that mimics collagen fibre arrangement
and zonal tensile strength of articular cartilage.
Acknowledgements. The authors acknowledge the
Medical Engineering Solutions in Osteoarthritis Centre of
Excellence funded by the Wellcome Trust and EPSRC.
Keywords. anisotropic scaffold; electrospinning; zonal
organization
Figure 1: Sequential electrospinning generated a bulk
material with different fibre arrangements and
morphologies, and depth-dependent tensile properties
similar to those of articular cartilage.
(8.O2) INFLUENCE OF CONDITIONED MEDIUM OVER THE
CHONDROGENIC DIFFERENTIATION OF ADULT STEM
CELLS IN 3D CO-CULTURES WITH ARTICULAR
CHONDROCYTES
Alves da Silva MA (1,2), Costa-Pinto AR (1,2), Correlo V
(1,2), Sol P (1,2), Bhattacharya M (3), Faria S (4), Reis RL
(1,2), Neves NM (1,2)
1. 3B´s Research Group, University of Minho, Portugal; 2.
IBB – Institute for Biotechnology and Bioengineering,
Portugal; 3. Department of Biosystems Engineering,
University of Minnesota, USA; 4. CMAT, University of
Minho, Portugal
Aim. Soluble factors released by chondrocytes have been
shown to influence stem cells differentiation onto the
chondrogenic lineage. Using conditioned medium
obtained from chondrocytes for stimulating stem cells
chondrogenic differentiation may be a very interesting
alternative for clinical application of these cells.
Therefore, we tested the influence of conditioned
medium obtained from articular chondrocytes cultures to
determine its influence on indirect co-cultures of human
bone marrow-derived MSCs (hBMSCs) and human
Wharton´s jelly MSCs (hWJSCs) seeded in 3D porous
scaffolds.
Method. Indirect co-cultures (using conditioned medium
obtained from a culture of human articular chondrocytes)
hBMSCs and hWJSCs were established. Cells were
isolated from human samples collected at São Marcos
hospital, under donors informed consent. Co-cultures
were performed in 3D fibrous and porous scaffolds,
composed by a blend of 50/50 chitosan and poly
(butylene succinate) – CPBS. Co-cultures were maintained
during 28 days.
Results. Both types of stem cells were able to undergo
chondrogenic differentiation. By the end of the
experiment co-cultures showed glycosaminoglycans
(GAGs) accumulation and up-regulated expression of
cartilage-related gene, for both types of adult MSCs
tested. The hWJSCs showed higher chondrogenic
differentiation ability when compared to hBMSCs, as
denoted by the higher values for GAGs accumulation and
cartilage–specific gene expression.
Conclusions. Using conditioned medium obtained from
articular chondrocytes induced the chondrogenic
differentiation of MSCs and ECM formation. The obtained
results showed that this new strategy enables the
development of new therapies for cartilage repair.
Keywords. Conditioned media, co-cultures, stem cells,
chondrocytes
(8.O3) IN VIVO EVALUATION OF A NOVEL
OSTEOCHONDRAL SCAFFOLD FOR OSTEOCHONDRAL
DEFECT REPAIR
Levingstone T (1), Schepens A (1), Thompson E (1),
Matsiko A (1), O’Brien F (1), Gleeson J (1)
1. Royal College of Surgeons in Ireland
Introduction. Osteochondral tissue has a complex layered
structure, organised into cartilage, calcified cartilage and
subchondral bone regions. It has poor regenerative
capacity and as a result over 15 million people worldwide
suffer from knee joint failure each year due to cartilage
breakdown (Frost and Sullivan, 2009). Current treatment
methods
include
drilling,
microfracture,
and
osteochondral grafting; however, no treatment has
managed to repair large osteochondral defects with longlasting hyaline cartilage (Klein et al, 2009). The aim of this
study was to evaluate the in vivo regenerative potential
of ChondroColl, a recently developed, patented
multilayer scaffold for osteochondral repair.
Methods. Collagen-based multi-layer scaffolds were
fabricated using a novel ‘iterative layering’ freezedrying
technique (WO2010084481). The in vivo performance
was evaluated using a rabbit medial femoral condyle
model. Scaffolds were implanted into 3mm diameter x
5mm depth critical sized defects. Repair tissue was
evaluated 12 weeks post implantation using micro-CT and
histological analysis.
Results. Macroscopic analysis at 12 weeks post
implantation showed a greater degree of tissue formation
in the scaffold group than the empty defect controls.
Repair tissue appeared to integrate well with surrounding
tissue with no signs of debris or inflammation (Fig. 1c).
The International Cartilage Repair Society (ICRS) scoring
system indicated the formation of significantly better
quality repair tissue in the scaffold implanted group.
Micro-CT (Fig. 1d) showed greater repair in the scaffold
group than the control, with evidence of subchondral
bone repair within the defect and formation of an
overlying cartilaginous layer. Histological analysis is
currently ongoing.
Conclusions. In vivo analysis of the novel multi-layer
scaffold showed that the scaffold enabled successful
generation of de novo bone and cartilaginous repair
tissue in the defect space. Further histological analysis is
on-going to evaluate level of cartilaginous healing.
Acknowledgements.
Enterprise
Ireland
Commercialisation Fund, Proof of Concept (PC/2007/331)
and Technology Development Phase (CFTD/2009/0104).
Keywords. Osteochondral, tissue engineering, cartilage
(8.O4) FIBRIN SCAFFOLD WITH GROWTH FACTORSENRICHED NANOFIBERS ENHANCED OSTEOCHONDRAL
REGENERATION IN MINIATURE PIGS
Filova E (1), Rampichova M (1), Vajner L (2), Lytvynets (1),
Mickova A (1), Martinova L (3), Motlik J (4,5) Uhlik J (4,5),
Amler E (4,5)
1. Institute of Experimental Medicine of the ASCR, Prague,
Czech Republic (CR); 2. Institute of Biophysics, 2nd Faculty
of Medicine, Charles University in Prague, CR; 3. Textile
Faculty, Technical University of Liberec, Liberec; 4.
Institute of Animal Physiology and Genetics of the ASCR,
Liběchov, CR; 5. Institute of Biophysics, 2nd Faculty of
Medicine, Charles University in Prague
Introduction. Nanofibers possess a high surface area
which enables adhesion of bioactive substances. The aim
of the study was to examine the effect of growth factorsenriched PVA nanofibers on the viability of mesenchymal
stem cells (MSC) in vitro and, subsequently, cartilage
regeneration in minipigs using fibrin scaffold containing
growth factors-enriched nanofibers.
Methods. PVA nanofibers were incubated with basic
fibroblast growth factor and insulin, and subsequently
seeded with MSC. The cell viability was examined using
MTT test after 1, 3, and 7 days. Same scaffold without
cells were mixed with Tissucol® and implanted into eight
load-bearing osteochondral defects in minipigs. As a
control, the defects in the left knees were left untreated.
Animals were sacrified 12 weeks after the surgery, and
evaluated histologicaly.
Results. The cell viability was significantly higher on
modified scaffold compared to pure PVA. In the animal
study, the scaffold group showed a regular formation of
isogenic lines of chondrocytes near the defect bases and
differentiation towards hyaline cartilage. Fibrocartilage
was found on the defect surface. The middle and basal
zones were predominantly alcian blue positive. Type II
collagen was positive in the non-cellular transient zone in
the newly formed cartilage and on the border of young
isogenic groups. In a control group, fibrocartilage or
unorganized fibrous tissue with isogenic groups of
chondrocytes was situated at the borders; fibrous tissue
accompanied by vascularization was observed on the
surface. Alcian blue was positive in the upper part of
defects; type II collagen was positive in the newly formed
cartilage.
Conclusions. The composite scaffold supported the
hyaline cartilage formation, therefore, the scaffold is
suitable for cartilage regeneration.
Supported by Grant Agency of AS CR grant No.
IAA500390702, MSMT CR grants No. 1M0510
(1M6798582302) and NPV II 2B06130, AV0Z – ASCR, No.
AV0Z50390512 and AV0Z50390703, Grant Agency of
Charles University No. 119209.
Keywords. nanofibers, cartilage, growth factors
(8.O5) CELLS FROM SYNOVIAL FLUID: SOURCE OF
AUTOLOGOUS
CELLS
FOR
CARTILAGE
TISSUE
ENGINEERING?
Maillard N (1), Grybek V (2), Merceron C (2), Portron S (2),
Lesoeur J (2), Masson M (2), Weiss P (2), Guicheux J (2),
Vinatier C (2)
1. Inserm U 791, LIOAD, group “STEP”, Nantes, France.
Pharmacy Department, University Hospital, Nantes,
France; 2. Inserm U 791, LIOAD, group “STEP” (Skeletal
Tissue Engineering and Physiopathology), Nantes, France
Introduction. Tissue engineering using mesenchymal
stem cells (MSC) for the treatment of cartilage defects
appears promising. Among the different sources of MSC
used in cartilage engineering such as bone marrow,
adipose tissue or synovial membrane, it remains difficult
to clearly identify the most clinically relevant source.
Recently, the presence of adherent cells in pathological
synovial fluid (SF) has been described. Given that SF is
easily accessible by simple joint puncture, the aim of this
work was to determine whether adherent synovial fluid
cells (ASFC) could represent an autologous cells source for
future applications in cartilage regeneration.
Materials
and
Methods.
Human
ASFC
isolated from synovial fluid puncture were characterized
for their (i) ability to form colony by CFU-F assay, (ii)
surface markers expression by flow cytometry and (iii)
multipotency.
For adipogenic
and
osteogenic
differentiation, cells were
cultured
in
specific
differentiation medium in monolayer for 14 and 28 days
respectively.
To
chondrogenically
differentiate
ASFC, cells were cultured in specific medium during 28
days in pellets. Cell differentiation was monitored at the
level of γmRNA by real time-PCR (ALPL, RUNX2, COL1A1,
COL2A1, ACAN, SOX9, COMP, PPAR). Alkaline
phosphatase (ALP) activity, histology (oil red O, alizarin
red) and immunodetection (type II collagen) were
performed.
Results. Our data show that ASFC exhibited proliferation
and colony-forming abilities. ASFC also expressed typical
stem cell surface markers. Additionally, they were able to
differentiate towards the chondro-, osteo- and
adipogenic lineages.
Discussion and Conclusions. These results show that
ASFC express some of the major MSC characteristics.
Wether ASFC could be able to promote cartilage
regeneration in adapted animal models should be paid
further attention.
Keywords. Synovial fluid cells, cartilage tissue
engineering.
(8.O6) THE USE OF FIBROBLASTS FOR THE
RECONSTRUCTION OF ANTERIOR CRUCIATE LIGAMENT:
RESEARCH ON THE SHEEP ANIMAL MODEL
López-Alcorocho JM (1), Guillén-García P (1), RodríguezÍñigo E (1), Guillén-Vicente I (1), Val-Garijo D (2), GuillénVicente M (1), Caballero-Santos R (1), García-Gómez F (1),
Fernández-Jaén T (1), Arauz S (1), Abelow S (1)
1. Clinica CEMTRO; 2. Hospital Carlos III
Summary. The rupture of the anterior cruciate ligament
(ACL) is currently treated with a surgical procedure that
implies the use of different tendons or ligaments to
reconstruct the damaged ACL. Currently, the research on
this field is focused in finding a new method to reduce the
time of recovery which with these techniques is now of 68 months. We have investigated the use of fibroblasts for
the reconstruction of broken ACL.
Material & Methods. Ten female sheep with a similar age
will be included in this study and were divided into 2
groups.
- Group A: Implanted with 5 million fibroblasts embedded
in the membrane
- Group B: Implanted with the membrane without cells
The animals undergone 2 surgeries: one surgery to take
an ACL biopsy and the other one to break the ACL and
implant the membrane with (Group A) or without (Group
B) cells.
After 3 months, the animals will be sacrificed and samples
from the ACL regeneration and from healthy areas
(control) will be taken. Histological and molecular studies
will be performed to compare both treatments between
them and with the control.
Results. The architecture of normal ACL was not
conserved either in the ACL treated with the membrane
with or without cells. However a high number of cells,
similar to fibroblast was found in the cell-treated ACL
than in those treated only with the collagen membrane,
indicating that probably these cells migrated from the
membrane to the damaged ACL. RT-PCR studies
performed demonstrated that these cells expressed type I
collagen, tenascin-C and MMP-13; indicating the
fibroblastic origin of the cells.
Conclusion. We think that this novel technique could be a
promising tool to treat the ACL rupture and represents a
first step in the use of tissue engineering for treatment of
the ACL rupture.
Keywords. ACL rupture, biomaterials
(8.O7) TREATMENT OF CHONDRAL DEFECTS WITH
AUTOLOGOUS CHONDROCYTES OR MESENCHYMAL
CELLS ON TYPE I/III COLLAGEN MEMBRANES IN THE
OVINE MODEL
Rodríguez-Íñigo E (1), Guillén-García P (1), LópezAlcorocho JM (1), Guillén-Vicente M (1), Caballero-Santos
R (1), Guillén-Vicente I (1), Santos-Molina E (1), GarcíaGómez F (1), Fernández-Jaén T (1), Arauz S (1), Abelow S
(1)
1. Clinica CEMTRO, Madrid, Spain
Introduction. Autologous chondrocyte implantation (ACI)
combined with a periosteal flap, was first performed in
the human knee in 1994. In MACI implants, chondrocytes
are seeded in a collagen I/III membrane functioning as
cell carrier. Some research has been focused in
developing techniques based on cell therapy using other
cells as mesenchymal cells (MSC).
Materials & Methods. Five 2-3 years-old female sheep
were included. A full 10 x 10 mm incision was made in the
articular cartilage of the medial femoral condyle. This
sample was used as a source o chondrocytes. A second
lesion of the same size was done at the trochlea. In this
lesion, microperforations were done. A sample of adipose
tissue from the Hoffa’s fat pad was taken to isolate MSC.
One and 5 million of cultured chondrocytes and 5 million
MSC, respectively, were seeded on a collagen I/III
membrane and then they were implanted. After 12 weeks
the animals were sacrificed and tissue samples in the
following areas were taken: a) MSC implant area, b)
microperforations area, and c) healthy tissue near of
perforation area. Histological and molecular studies were
carried-out made by hematoxilin-eosin and safranin-O
staining. Relative expression of aggrecan and types I and
II collagens was determined by real-time polymerase
chain-reaction.
Results. The tissue architecture and the expression
pattern of proteoglycans was more similar to that
observed in normal cartilage in the lesions treated with 5
million chondrocyte followed by 1 million and by MSC and
microperforations. These results were supported with the
studies of gene expression.
Conclusion. The implantation of 5 million of cultured
autologous chondrocytes on I/III collagen membranes
seems to give better histological and molecular results
than 1 million cells. Microperforations and Hoffa’s fat pad
derived MSC seem to have no role in the reparation of
damaged cartilage.
Keywords. Cartilage repair, ovine model, collagen
membrane
(8.O8) TOWARDS IN SITU THERAPY OF OSTEOARTHRITIS:
CARTILAGE SPECIFIC CHEMOKINES AND THEIR ROLE IN
HUMAN MESENCHYMAL STEM CELL MIGRATION
Biens K (1), Dehne T (1), Karlsson C (2), Lindahl A (2),
Sittinger M (3), Ringe J (3)
1. Tissue Engineering Laboratory and Berlin-Brandenburg
Center for Regenerative Therapies, Charité University
Medicine, Berlin, Germany; 2. Institute of Laboratory
Medicine, Department of Clinical Chemistry and
Transfusion Medicine, Sahlgrenska University Hospital,
Gothenburg, Sweden; 3. Tissue Engineering Laboratory
and Berlin-Brandenburg Center for Regenerative
Therapies, Charité University Medicine, Berlin, Germany
Introduction. In situ Tissue Engineering represents a
current approach for the regeneration of damaged or
diseased joint tissues implying the use of supportive
bioscaffolds and bioactive molecules promoting
recruitment of mesenchymal stem cells (MSC) and their
subsequent differentiation. Several studies have
designated chemokines (Ck) as ideal candidates for MSC
attraction. Further, it was shown that human articular
cartilage secretes Ck that might be potent to attract MSC
in vivo. Thus our studies focussed on characterisation of
differentially expressed Ck in Osteoarthritis (OA) and
healthy cartilage and their ability to induce chemotaxis in
MSC.
Methods. Human articular cartilage biopsies were
collected from donors with macroscopical and
microscopical signs of OA as well as donors with intact
cartilage. RNA was isolated from the biopsies and
subjected to whole genome microarray analysis. In
addition, cartilage and chondrocyte conditioned
supernatants were collected and analysed for their Ck
profile using protein arrays. Attraction impact of
supernatants on human MSC from healthy and OA donors
was examined in 96-well chemotaxis assays.
Results. Among other several new marker genes,
microarrays revealed an increased expression of the Ck
CXCL2, CXCL3, CXCL14, CCL3 and CCL4. Proteomics
confirmed the OA specific secretion of CXCL2-3 and
migration assays demonstrated a significantly higher
recruitment of MSC by OA cartilage derived supernatants.
Conditioned medium from OA chondrocytes displayed
increased secretion levels of CXCL1-3, CXCL8 and CCL2.
However, no increase in recruitment of MSC was
detected here.
Conclusion. Our results show OA cartilage specific gene
expression and release of Ck and their potency to recruit
MSC from healthy and OA donors. Here, increased levels
had either stimulating or inhibiting effects on MSC
attraction, displaying involvement of more complex
regulations. In conclusion, these are revealing findings
towards a Ck guided in situ therapy of OA using MSC.
Keywords.
osteoarthritis;
chemokines;
in
situ
regeneration; mesenchymal stem cells
(8.O9) A SELF-SETTING HYDROGEL MECHANICALLY
REINFORCED WITH A MARINE EXOPOLYSACCHARIDE AS
A SCAFFOLD FOR CARTILAGE TISSUE ENGINEERING
Rederstorff E (1,2), Weiss P (1), Sourice S (1), ColliecJouault S (2), Fellah B (1), Masson M (1), Guicheux J (1),
Vinatier C (1,3)
1. IFREMER/LIOAD; 2. INSERM/LIOAD; 3. GRAFTYS
Polysaccharides-based hydrogels have been widely used
as 3D scaffolds for cartilage tissue engineering. However
none of them showed both mechanical and biological
adequate properties. To develop a biomechanically and
biologically competent hydrogel for cartilage tissue
engineering, a cellulose-based hydrogel (Si-HPMC) was
reinforced with a marine exopolysaccharide called GY785.
Previously, we have shown that GY785 EPS addition has
improved the mechanical properties of the Si-HPMC.
Therefore, the aims of the present work were (i) to
investigate the ability of this Si-HPMC/GY785 to allow the
maintenance and the recovery of a chondrocytic
phenotype and (ii) to evaluate the potential of this SiHPMC/GY785 associated with chondrocytes to form a
cartilaginous tissue in vivo.
Primary rabbit articular chondrocytes (RAC) or
dedifferentiated RAC were cultured in 3D within SiHPMC/GY785 for 3 weeks. The chondrocytic phenotype
was investigated by real-time PCR (agrecan, type I and II
collagen), alcian blue staining (sulphated GAG) and
immunostaining (type II collagen). Finally, the ability of SiHPMC/GY785 to form a cartilaginous tissue was
investigated by in vivo transplantation of RAC and equine
nasal chondrocytes (EqNC) with Si-HPMC/GY785
subcutaneously in nude mice. After 3 weeks, implants
were histologically characterized to determine the
presence of sulphated GAG (Alcian blue) and type II
collagen (Immunostaining).
Our results showed that primary RAC 3D-cultured within
Si-HPMC/GY785 expressed type II collagen and agrecan
after 3 weeks. These cells also produced an extracellular
matrix containing sulphated GAG and type II collagen.
When dedifferentiated RAC were replaced in 3D within SiHPMC/GY785 the expression of type II collagen and
agrecan were recovered and type I collagen expression
was decreased. Finally, histological analysis of hybrid
constructs transplanted in nude mice revealed the
production of sulphated GAG and type II collagen. This
study
indicates
that
mechanically
GY785
exopolysaccharides reinforced Si-HPMC could appear as a
promising hydrogel for cartilage tissue engineering.
Keywords. Cartilage, hydrogel, tissue engineering
(8.O10) IS SELF ASSEMBLY USING PROGENITOR CELLS A
BETTER APPROACH TO ENGINEERING FUNCTIONAL
CARTILAGE TISSUE THAN HYDROGEL ENCAPSULATION?
Mesallati T (1), Buckley CT (1), Kelly DJ (1)
1. Trinity Centre for Bioengineering, School of Engineering,
Trinity College Dublin, Ireland
Introduction. Agarose encapsulation and self assembly
are two promising methods that have been proposed to
engineer functional cartilage tissue. The objective of this
study was to compare the functional properties of
cartilaginous tissues engineered using Infrapatellar Fat
Pad (IFP) derived MSC’s using either agarose
encapsulation or self assembly.
Methods. Porcine fat pad derived MSC’s were
encapsulated in agarose, forming cylinders of either
1.5mm or 3mm thickness. Two seeding densities were
examined for each thickness of gel (0.88E6 or 4E6 total
cells), resulting in four overall gel seeding densities (15E6,
30E6, 68E6, and 136E6 cells/ml). Self assembled
constructs were formed by adding either 0.88E6 or 4E6
cells in chemically defined media (CDM) between PDMS
O-rings. Constructs were maintained for the first 3 weeks
in CDM supplemented with TGF-β3, upon which TGF-β3
supplementation was either withdrawn (TGF-) or
maintained (TGF+) for a further 3 weeks.
Results. Matrix accumulation was greater for higher
seeding densities (4E6 cells) using both methodologies
(Fig.1). Within the low seeding density group we observed
greater sGAG accumulation in agarose gels compared to
self assembled constructs (TGF+), however at high
seeding densities, self assembled constructs (TGF-) were
comparable to agarose groups (TGF+). Collagen
accumulation was greater in the agarose constructs
(TGF+) compared to the corresponding self assembly
groups.
Conclusions. In general we observed greater matrix
accumulation in agarose constructs compared to self
assembly, perhaps indicating it as the more desirable
method of the two. However, when normalised to tissue
wet weight (data not shown), matrix accumulation was
greater in the lighter self assembled constructs,
approaching values seen in normal articular cartilage. This
suggests that self assembly results in the development of
more functional cartilaginous constructs.
Acknowledgements. Funding was provided by IRCSET and
an SFI President of Ireland Young Researcher Award
(08/Y15/B1336).
Keywords. Self-assembling process; Agarose hydrogel;
Functional tissue engineering; mesenchymal stem cells
(8.O11) NATURAL CHITIN MATRICES, ISOLATED FROM
MARINE SPONGES, AS SUITABLE 3D-SCAFFOLDS FOR
CARTILAGE TISSUE ENGINEERING
Steck E (1), Hoffmann M (1), Ehrlich H (2), Richter W (1)
1. Orthopaedic Universtity Hospital, Research Center for
Experimental
Orthopedics,
Universitätsklinikum
Heidelberg, Germany; 2. Institute of Bioanalytical
Chemistry, Dresden University of Technology, Germany
Introduction. Tissue engineering (TE) of articular cartilage
is based on a suitable 3D-scaffold. Promising results were
reported for synthetic chitosan (chitin-derivative) based
scaffolds. Marine sponges of the genius Verongida posses
a naturally developed 3D-chitin-skeleton that has been
optimized by evolution to support cell seeding and
nutrient supply. Aim of this study was to characterise this
biomaterial regarding biocompatibility and support of a
cartilage-like extracellular matrix (ECM) deposition.
Methods. Chitin scaffolds were isolated from Aplysina
cauliformis by repeated extraction of other constituents
with acidic acid and NaOH. For in vitro analyses porcine
articular chondrocytes were cultured in the scaffolds in
chondrogenic medium. For in vivo analyses human
chondrocytes were seeded into the scaffolds and
implanted subcutaneously into SCID-mice. Samples were
analysed for cell vitality and by histological staining. To
discriminate between donor and host cells an in-situhybridization protocol was developed specifically
detecting human and mouse genomic repetitive
elements.
Results. Stability and handling of the chitin scaffolds were
excellent, no destruction was observed during cell
seeding, cultivation, or transplantation. In vitro, primary
cells were distributed throughout the scaffold
accompanied by high cell vitality (> 80%). After 4-6 weeks
cells synthesized a cartilage-like ECM as determined by
alcian-blue and type-II-collagen staining. In situ
hybridization demonstrated that exclusively implanted
human chondrocytes deposited a cartilage-typical ECM
and no cells dedifferentiated or evaded into the
surrounding fibrous mouse tissue. A small number of
murine cells (<5%) were found inside the proteoglycanrich cartilage matrix which might have invaded the
regenerate before deposition of the cartilage-like ECM.
Conclusion. The natural chitin scaffolds represent a
promising 3D-matrix for cartilage TE. The structure would
particularly be suitable for targeted chemical
modifications allowing the specific upgrading with factors
supporting cell migration, adhesion, proliferation, or
chondrogenic differentiation when replacement of
chondrocytes by progenitor cells or in situ cartilage repair
strategies are envisaged.
Keywords. marine chitin sponges, cartilage tissue
engineering, extracellular matrix, species-specific cell
detection
(8.O12) STEM CELL SURFACE MARKER SSEA-4 SELECTS
FOR
CHONDROPROGENITORS
WITH
ENHANCED
CHONDROGENIC POTENTIAL IN CULTURED HUMAN
ARTICULAR CHONDROCYTES
Schrobback K (1), Wrobel J (1), Hutmacher DW (1),
Woodfield T (2), Klein TJ (1)
1. Institute of Health and Biomedical Innovation,
Queensland University of Technology, Brisbane, Australia;
2. Department of Orthopaedic Surgery, University of
Otago Christchurch, New Zealand
Introduction. One important challenge for cartilage tissue
engineering is to produce a clinically relevant number of
cells with consistent chondrogenic potential. In vitro
expansion of autologous chondrocytes results in a
heterogeneous population of dedifferentiated cells and
variable amounts of chondroprogenitors. Identification
and isolation of chondroprogenitors could lead to more
consistent cartilage formation. We have found that
subpopulations of cultured human articular chondrocytes
express SSEA-4, a cell surface marker of embryonic and
mesenchymal stem cells. In this study, we characterised
the proliferation and differentiation potential of human
chondrocytes sorted according to SSEA-4 levels.
Methods. Articular cartilage was obtained from three
consenting patients undergoing limb amputations.
Isolated chondrocytes were expanded and SSEA-4 levels
were assessed over several passages by flow cytometry.
Cell populations either positive or negative for SSEA-4
were separated at passage 2 by fluorescence-activated
cell sorting and either propagated in monolayers for one
more week with DNA levels monitored every three days
or redifferentiated in pellet cultures over two weeks. In
differentiation cultures, pellet sizes were determined and
expressions of aggrecan, collagen II and I were quantified
by qRT-PCR.
Results. SSEA-4 was not detectable in freshly isolated
chondrocytes. However, SSEA-4 levels peaked at
66.7±4.4% positive cells after approximately five
population doublings and decreased thereafter. Cultured
chondrocytes sorted for SSEA-4 formed 25%±3.1% larger
pellets and expressed higher levels of chondrogenic
markers during redifferentiation than SSEA-4-negative
chondrocytes. However, the latter proliferated slightly
faster (1.12±0.1 days doubling time) in monolayers than
SSEA-4 expressing cells (1.29±0.1 days) (p<0.05).
Conclusions. Our observations indicate that the stem cell
surface antigen SSEA-4 can be used to select for
chondroprogenitors with enhanced chondrogenic
differentiation capacity in cultured human chondrocytes.
Future research will be focussed on the cellular
characterisation of purified SSEA-4-positive cells to
confirm their superior chondrogenic potential in vivo.
Keywords. cartilage, tissue engineering, surface marker
(8.O13) THE ROLE OF CELLULAR COMMUNICATION IN
BONE
MARROW
DERIVED
STROMAL
CELL
CHONDROGENIC DIFFERENTIATION
Potier E (1), Rivron N (2), Van Blitterswijk C (2), Ito K (1)
1. Department of Biomedical Engineering, Eindhoven
University of Technology, Eindhoven, The Netherlands; 2.
Institute for Biomedical Technology and Technical
Medicine, University of Twente, Enschede, The
Netherlands
Bone marrow-derived stromal cells (BMSCs) are
envisioned as regenerative cells for numerous tissues,
including cartilage. Success of BMSC-based therapies,
however, relies on a number of methodological
improvements, among which is better understanding and
control of their differentiation pathways. We investigated
here the role of cellular communication (through
paracrine signaling and/or cell-cell contact) in the
chondrogenic potential of BMSCs.
Bovine BMSCs (n=3 donors) were encapsulated in alginate
beads as dispersed cells at 3, 7, and 14 millions cells/ml
and as micro-aggregates at 7 millions cells/ml thus
creating different paracrine signaling and cell-cell contact
conditions. BMSCs were cultured for 21 days under
hypoxia (2%O2) and TGFb3 stimulation (10ng/ml). At d0
and d21, cell phenotype was characterized by RT-qPCR
(type I and II collagens, sox9, aggrecan, TGFb); produced
matrix by histology (Alcian blue staining) and biochemical
assays (glycosaminoglycan (GAG) and DNA content); cell
morphology by histology (phalloidin staining); and cell
viability by live/dead staining.
In all conditions, BMSCs stayed viable and DNA content
remained constant up to 21 days. Major chondrogenic
markers (type II collagen, aggrecan, sox9) were clearly upregulated at day 21, with a higher up-regulation for
dispersed cells (Figure). Matrix production (GAG/DNA
content) increased in time but without significant
differences between groups (Figure). Histological analysis
is under progress. This study showed that, under TGFb
stimulation and in the range of cell concentrations used
here, endogenous paracrine signaling does not
significantly affect BMSC chondrogenic differentiation, as
all dispersed conditions led to the same outcomes. Cellcell contact (micro-aggregates) has a negative effect on
chondrogenic marker expression that is not reflected at
the matrix level. Endogenous paracrine signaling and cellcell contact, however, may have a greater impact on
BMSC chondrogenic differentiation under other
stimulants such as mechanical loading which may rely on
endogenously produced factor or cell-cell communication
for amplification of their effects.
Keywords. Bone marrow-derived stromal cells;
chondrogenic differentiation; paracrine signaling; cell-cell
contact
(8.O14)
PERIOSTEAL
FLAP
SUBSTITUTE
FOR
AUTOLOGOUS CHONDROCYTE IMPLANTATION
Tai BCU (1), Du C (1), Wan ACA (1), Ying JY (1)
1. Institute of Bioengineering and Nanotechnology
Autologous chondrocyte implantation (ACI) is one of the
options available to treat osteoarthritis. In this procedure,
a periosteal flap is harvested and secured over the defect
site to hold the implanted chondrocytes in place.
However, the use of the graft is often associated with
graft hypertrophy and an increase in subchondral bone
density. Hence, a synthetic substitute is highly desirable.
In this study, we have a developed a PVA-based
membrane to address the problems associated with the
use of the periosteal flap. The membrane displayed good
mechanical properties, with a Young’s modulus of about
1MPa – above the minimum required for hyaline
cartilage. Modification of the membrane to present the
integrin-binding peptide, RGD, improved initial cell
attachment by up to 4-fold, pointing towards improved
chondrocyte survival in vivo. In vitro culture of bone
marrow-derived human mesenchymal stem cells (hMSCs)
revealed that the cells remained attached and viable on
the membranes for up to 2 months. Gene expression
studies for bone markers, namely collagen type I, RunX2
and bone sialoprotein (BSP), of hMSCs cultured on the
membranes showed lower expression as compared to
hMSCs cultured on tissue culture plastic, thus lowering
the risk of graft hypertrophy. In vivo implantation of the
membrane material showed good biocompatibility. These
findings demonstrated that the RGD-modified PVA
membranes are a potential substitute for the periosteal
flap used in ACI, as well as other applications in which the
periosteum is required.
Keywords. Biomaterials, Membrane, Cartilage
(8.O15) COMPRESSIVE BIOMECHANICAL PROPERTIES OF
A NEW BIO-COLLAGEN SCAFFOLD FOR CARTILAGE
TISSUE ENGINEERING
Elsaesser AF (1), Schwarz S (1), Koerber L (2), Seitz A (3),
Duerselen L (3), Breiter R (2), Rotter N (1)
1. Department of Otorhinolaryngology, University Medical
Center Ulm; 2. Department of Medical Bio-Technology,
University Erlangen; 3. Institute of Orthopedic Research
and Biomechanics, University of Ulm
Introduction. Defects of cartilage in nose and ear are
frequent problems caused by trauma or cancer. The need
for biomaterials for reconstruction of auricle or nasal
septum therefore is enormous. A newly developed biocollagen scaffold from decellularised porcine cartilage
shows properties more promising than the materials
currently in use for tissue engineering applications. The
aim of our study was to analyse this novel material in
combination with human chondrocytes.
Methods. The proportion of glycosaminoglycans was
measured with a DMMB assay, the amount of collagen
with a hydroxyprolin assay. To show effects of cellular
immigration, scaffolds were seeded with primary human
nasal septal chondrocytes up to 42 days in chondrogenic
differentiation medium.
Uniaxial confined compression tests were conducted to
determine and compare the mechanical properties of
native and processed scaffolds (n=12 each). Progress of
seeding and immigration of chondrocytes were analysed
with histological and immunohistochemical staining.
Results.Due to the decellularisation process the apparent
modulus of the scaffolds decreased from 6.5 ± 2.3 MPa to
2.2 ± 1.2 MPa. The DMMB assay showed that the content
of glycosaminoglycans was significantly reduced. Relating
to the dry weight the proportion of collagen increased,
while the fraction of denatured collagen changed from
approximately 25 % in native porcine nasal septal
cartilage to 50 % in the processed scaffold.
Scaffolds seeded with human septal chondrocytes
regained stability. Cells started to produce and
incorporate aggrecan into the scaffold in less than 7 days.
Conclusion. Decellularisation and removal of noncollagenous components of extracellular matrix from
porcine nasal septal cartilage leads to changes in matrix
properties in vitro. Even though the resulting scaffolds
maintained their shapes with sufficient mechanical
stability and could therefore be suitable for surgical
applications. We expect that after seeding with
chondrocytes and implantation in vivo the cartilage
constructs could retrieve full stability and function.
Keywords. Cartilage reconstruction, biomatrices, human
chondrocytes
(8.O16) RECONSTRUCTION OF THE AURICLE WITH THE
USE OF BACTERIAL CELLULOSE
Feldmann EM (1), Sundberg JF (2), Schwarz S (1),
Gatenholm P (2), Rotter N (1)
Center Ulm; 2. Chalmers University of Technology; 3.
Department of Otorhinolaryngology, University Medical
Center Ulm
Introduction. Porous bacterial cellulose (BC) is a
promising new nano-biomaterial which has shown to
possess impressive biomechanical properties and
excellent biocompatibility for the use in the field of tissue
engineering. BC has already been used as biomedical
implant in the field of blood vessels, skin and meniscus
replacements. For tissue engineering of an auricle no
suitable materials have been found until today. BC seems
to be a promising candidate.
Methods. Three-dimensional BC scaffolds are synthesized
by the bacterium Gluconacetobacter xylinus. During the
fermentation process incorporated paraffin wax beads
form interconnected micro-pores. Human chondrocytes
isolated from auricular, septal and rib cartilage have been
expanded and seeded in different densities onto these
scaffolds and cultivated for up to 5 weeks. Adhesion,
distribution, proliferation and production of extracellular
matrices have been detected with histological and
immuno-histological staining methods as well as RT-PCR.
Results. Human chondrocytes adhere at the BC scaffolds
and migrate into the interconnected pores where they
produce their own cartilage specific extracellular matrix
proteins such as collagen II and aggrecan. We found that
all cell types are able to retain their differentiated
phenotype in this three-dimensional culture system.
Furthermore cells proliferate and generate a thick matrix
layer on the surface of the BC. Yet, the homogeneous
distribution of the chondrocytes in the material is
restricted due to uneven interconnectivity of the pores.
Conclusions. The experiments show that human
chondrocytes adhere and spread within porous bacterial
cellulose while no cytotoxic effects are detectable. BC
seems to be a suitable material for the cultivation of
human chondrocytes. Continuing experiments for the
production of auricular shaped customizable 3D BC
scaffolds and the advancement of interconnectivity of the
pores are in progress.
Acknowledgement. Supported by the 7th framework
programme the EU – Euronanomed - programme EAREG
Keywords. ear cartilage, reconstruction, tissue
engineering
(8.O17) CARTILAGE TISSUE REPAIR FROM CLINICAL AND
BIOMATERIALS
PERSPECTIVE:
DECELLULARIZED
CARTILAGE AS A NOVEL BIO-MATRIX
Schwarz S (1), Elsaesser AF (1), Koerber L (2), Breiter R (2),
Rotter N (1)
Center Ulm; 2. Department of Medical Bio-Technology,
University Erlangen
Introduction. Damage or malformation of cartilage
structures in the head and neck region are often caused
by trauma, tumor resection or congenital defects.
New allogenic and xenogenic collagen bio matrices could
be the solution for several problems in reconstruction like
multistage surgeries, donor site morbidity, inflammatory
reactions or extrusions. These bio implants from natural
origin offer a high versatility and good mechanical
properties, making them interesting candidates for many
Materials and Methods. Applying a chemical process,
human, porcine and rat nasal septum cartilage samples
were completely decellularized and sterilized.
Scaffolds were preincubated for 24 h and seeded with
1x106 primary nasal chondrocytes (PNC). After cell
adhesion scaffolds were transferred individually to new
wells and cultivated for up to 42 days to examine
biomaterials biocompatibility, cytotoxicity and migration
behavior of human PNC. Histological as well as
immunohistochemical stainings were performed.The
vitality of the cells was measured using MTS assay and
PI/FDA staining.
Results and Discussion.We performed in vitro allogenic
and xenogenic models by seeding processed cartilage biomatrices from different species with human PNC. Human
cells adhere on scaffolds and infiltrate the matrix. Cells
occupied empty lacunae independently of the original
species of the processed tissue. Two weeks after seeding
scaffolds cells synthesized new ECM. At each time point
cell population was equal and approximately 1,52x105
cells per scaffold until day 42. The MTS assay showed the
intact metabolism of the cells and their vitality. No
cytotoxic effects could be detected.
Conclusions. The processed cartilage matrices are
completely sterile, free of cells and proteoglycans but are
still chondroconductive. No cytotoxic effects caused by
the process were detectable. In vitro the biocompatibility
between different species could be shown by allogenic
and xenogenic models. Therefore the application of
processed xenogenic and allogenic cartilage bio-matrices
in human applications seems to be possible.
Keywords. Cartilage, tissue engineering, biomatrices,
head and neck surgery
(8.O18) TIME COURSE OF JOINT CARTILAGE
REGENERATION
USING
POLY-ETHYL-ACRYLATE
SCAFFOLDS IN RABBITS
Sancho-Tello M (1), Martín de Llano JJ (1), Ruiz-Saurí A
(1), Gastaldi P (2), Forriol F (2), Monleón-Pradas M (3),
Gómez-Ribelles JL (1,3), Carda C (1,3).
1. Dept. Patología, Fac. Medicina y Odontología, Univ.
Valencia; INCLIVA; CIBER en Bioingeniería, Biomateriales y
Nanomedicina, Valencia, Spain; 2. Hospital de la
Malvarrosa, Valencia, Spain; 3. Center Biomaterials Tissue
Engineering, Univ. Politécnica Valencia; Centro Invest.
Príncipe Felipe, Regenerative Medicine; CIBER BBN
Valencia, Spain
Introduction. The aim of this work is to study the time
course of articular cartilage regeneration induced by
tissue engineering techniques in experimental animals.
Methods. A 3-mm diameter full depth chondral defect
was produced in the rabbit knee joint, injurying
subchondral bone in order to allow blood to flow into the
defect site. A biostable scaffold was laid to fit into the
chondral defect. The scaffold were made of a poly(ethyl
acrylate-co-hydroxyethyl acrylate) copolymer with 90% of
ethyl acrylate monomeric units, containing a well
interconnected spherical pores (mean diameter of 90
microns). Scaffolds were allowed to swell with the animal
blood that flowed from the defect. Tissue regeneration
was studied after 1, 2 weeks, 1, 3 and 12 months with
histological techniques.
Results. Regeneration started with initial activation of the
chondrocytes located near the edge of the excavated host
tissue, where 8-10 chondrocytes where observed in
several lacunae, 1 week after implantation. Besides,
incipient tissue formation was observed inside scaffold
pores, differentiated from mesenchymal cell arriving from
subchondral bone. One month after implantation, a well
formed layer of tissue was observed over the scaffold,
alligned with condylar surface. Thereafter, scaffolds were
shifted towards suchondral bone while they were invaded
by tissue filling their pores. After 3 months, excellent
tissue regeneration was obtained at the cartilage defect
site, with a well organized layer of hyaline cartilage at the
condylar surface. The pores were filled mostly with
cartillaginous tissue in its upper and central parts, and
bone tissue adjacent to the subchondral bone.
Conclusions. Synthetic scaffolds induced regeneration of
injured joint surface, while they were shifted from the
articular surface towards subchondral bone, while were
invaded by cells that formed neotissue within their pores.
Acknowledgements: Grant MAT 2007-66759-C03-01-03.
Key words. regeneration, articular cartilage, scaffold
(8.O19)
REGULATION
OF
OSTEOGENIC
AND
CHONDROGENIC DIFFERENTIATION OF MESENCHYMAL
PROGENITOR/STEM CELLS BY IL-1β AND OXYGEN
Mumme M (1), Wolf F (1), Jakob M (1), Wendt D (1),
Martin I (1), Barbero A (1)
1. Departments of Surgery and Biomedicine, University
Hospital Basel, Basel, Switzerland
Introduction. Because of their regenerative capabilities,
Bone Marrow-derived Mesenchymal Stem/Stromal Cells
(BM-MSC) are attractive for the repair of osteochondral
defects. The milieu of the damaged joint usually contains
many inflammatory cytokines, and is characterized by
variable oxygen percentages. This work aims at studying
the influence of interleukin-1β (IL-1β) and oxygen
percentage on the chondrogenic and osteogenic
differentiation of human BM-MSC in vitro.
Method. Expanded human BM-MSC (N=5 donors) were
cultured with different patho-physiological IL-1β
concentrations (0, 50 and 1000pg/ml) and oxygen
percentages (19%, 5%, 2%) for 3 weeks in 3D pellets with
a defined chondrogenic medium or in monolayer with
medium containing osteogenic factors. Bone marrow
aspirates were also cultured clonally to assess colony
forming unit osteoblast (CFU-O) and fibroblast (CFU-F).
Pellets were analyzed for Glycosaminoglycans (GAG) and
DNA amount, and by RT-PCR (Collagen II, X). Osteogenic
monolayers were analyzed for calcium accumulation and
by RT-PCR (Bone-sialoprotein, Osteocalcin, Indian
hedgehog) more reproducibly when used at low
concentration as evidenced by increased calcium
accumulation (2-fold), expression of all the osteogenic
genes and CFU-O/CFU-F ratio (1.2-fold), (ii) reduced at
low oxygen. ,βResults: Chondrogenic differentiation of
BM-MSC was (i) reduced under IL-1β 1000pg/ml as
evidenced by reduced amounts of GAG (5-fold) and
collagen II and X expression (2 order of magnitude), (ii)
moderately but reproducibly increased under IL-1β
50pg/ml, (iii) generally reduced at low oxygen.
Osteogenic differentiation was (i) enhanced by IL-1.
Conclusion. The results of this study indicate that the
exposure to low doses of Il-1β can enhance both the
osteogenic and chondrogenic differentiation potential of
BM-MSC in vitro. Controlling the inflammatory
environment could enhance the success of therapeutic
approaches
for
traumatic
and
degenerative
osteochondral lesions by resident MSC and as well
improve the engineering of implantable tissues.
Acknowledgment. We would like to acknowledge the
European Union for financial support (OPHIS; #FP7-NMP2009-SMALL-3-246373)
Keywords. osteochondral, mesenchymal progenitor/stem
cells, IL-1β, oxygen
(8.P1) NOVEL CULTURING TECHNIQUE CREATES CLINICAL
SIZED ARTICULAR CARTILAGE CONSTRUCTS
Khan AA (1), Surrao DC (2), Waldman SD (2)
1. University of Oxford, United Kingdom; 2. Queen's
University, Canada
Cartilage tissue engineering requires large cell numbers
for construct formation, which is a major limitation. Our
previous work demonstrated a continuous flow
bioreactor, with NaHCO3 supplemented media to
improve cell proliferation and ECM deposition, by
creating a near infinite supply of nutrients and by
buffering media. Hence, the aim of this study was to use
the above technique to produce clinical sized constructs
(> 1 cm2) without causing donor site morbidity (~300mg
with 2600 cells/mg).
The different seeding techniques used to engineer
constructs included: monolayer (20,000 cells or 666
cells/mm2), pellet (200,000 cells), biopsy (5mm diameter
constructs) and minced (5mm diameter biopsies cut into
smaller pieces). The constructs were cultivated in a
continuous flow bioreactor; with 14 mM NaHCO3
supplemented media, at a flow rate of 10 μL/min for 6
weeks, maintained at 37°C, 95% relative humidity and 5%
CO2. After 6 weeks of culture the tissue weight, thickness
and ECM deposition were determined.
Monolayer constructs outperformed all the other
constructs investigated in this study, while minced and
biopsy constructs recorded inconsistent data. Monolayer
and pellet constructs recorded the following values:
thickness: 2069 ± 90 and 1600 ± 47 µm, DNA: 199 ± 33
and 51.6 ± 17 µg/construct, GAG: 8908 ± 1089 and 3428 ±
458 µg/construct, and collagen: 2843 ± 150 and 1495 ±
272 µg/construct, respectively. This significant increase in
monolayer ECM accumulation could be due to the
combined effect of the bioreactor and NaHCO3
supplemented media. Additionally, the large surface to
volume ratio per cell in monolayer compared to the pellet
construct (chondrocytes though in larger numbers were
tightly packed together) could have provided the cells
greater accessibility to nutrients while allowing the
chondrocytes to divide, synthesize/accumulate ECM in
the monolayer without constraint.
Keywords. sodium bicarbonate; continuous flow
bioreactor; seeding techniques; extracellular matrix;
articular cartilage; chondrocytes; tissue engineering
(8.P2) CHITOSAN-PVA HYDROGELS AS A SCAFFOLD FOR
AURICULAR NEOCARTILAGE FORMATION. MECHANICAL
PROPERTIES CHARACTERIZATION
Velasquillo C (1), Garnica IM, (1), Vázquez N (1), SánchezArévalo FM (2), Martí nez V (1), García Z (3), Solis L (3),
Ibarra C (3), Luna-Barcenas G (3)
1. Tissue Engineering, Cell Therapy and Regenerative
Medicine Unit, National Institute of Rehabilitation; 2.
Materials Research Institute, UNAM; 3. Polymer &
Biopolymer Research Group Cinvestav Queretaro
Introduction. Tissue engineering (TE) of cartilage for
reconstructive surgery has proven to be a promising
option for the treatment of microtia and other disorders
involving cartilage deficiency. The goals of this study
were: 1. to determine cell and adhesion viability, and 2.
to measure mechanical properties of a biosynthetic
hybrid construct. Chitosan (CTS)-Poly (vinyl alcohol) (PVA)
films were tested as scaffold for auricular chondrocytes as
next step towards the clinical application of TE therapies
for pinna reconstruction.
Material and Methods. Auricular cartilage was obtained
from New Zealand rabbits. Cartilage was digested
mechanically and enzymatically. Biopolymers of CTS-PVA
were crosslinked with epichlorohydrin (ECH) to cast films
and were seeded with auricular chondrocytes and
cultured in standard in vitro conditions. Cell viability onto
the polymer was determined by calcein, and morphology
characteristics were studied by hematoxylin staining.
Environmental Scanning electron microscopy (ESEM) was
used to analyze cell adhesion to the polymer. Rabbit’s
cartilage and Q-PVA-ECH hydrogel were mechanically
characterized by uniaxial tension test.
Results. Cells seeded onto Q-PVA-ECH were viable and
showed chondral characteristics. Immunohistochemical
analysis tested positive for collagen II, aggrecan and
elastin. ESEM showed cell adhesion to the polymer. The
average ultimate tensile strenght (UTS) for the rabbit
cartilage, was 4.7 ± 1.6 MPa. The Young modulus for this
material was 45 ± 15 MPa. Based upon mechanical
properties characteristics, CTS-PVA-ECH hydrogels mimic
the human articular cartilage and it can be considered as
mechanically equivalent biomaterial.
Conclusion. Q-PVA-ECH polymer was successfully used to
engineer elastic cartilage and may have potential to be
used for reconstruction of the external ear.
Acknowledgements. We gratefully thank CONACYT
(78798 and 114359) for partial financial support.
Keywords. auricular cartilage, tissue engineering,
Mechanical properties, Chitosan-PVA, scaffold
(8.P3) IN VITRO EVALUATION OF COMPOSITE
CARBOXYMETHYLCELLULOSE (CMC) AND BICALCIUM
PHOSPHATES (BCP) IN ARTICULAR CARTILAGE REPAIR
Freitas DG (1), da Silva SN (1)
1. Center Federal Technological Education of Minas
Gerais, Brazil
The articular cartilage presents a structure anatomic
physiological complex with a thin layer viscoelástic tissue
aneural, avascular, aliphatic, anisotropic composed of
extracellular matrix populated cell discharge of weight
which is the ends of bone all joints sinoviais and that
allows smooth stable and smooth with minimal friction of
the areas of contact. The various strategies used in tissue
engineering as support for maintenance, proliferation and
differentiation of cells (chondrocytes and others) allowing
after trauma and/or diseases repair through the
formation of a new architecture (cell, extracellular matrix
and
irrigation)
of
cartilage.
Hydrogels
carboxymethylcellulose (CMC) and hydroxyapatite/betaphosphate tricálcio (BCP) has been studied as construct
for this application by their characteristics and rheologic
hydrophilic behavior appropriate macro and micro
mechanical this component anatomic. The objective of
this work was to evaluate the rheology and the use of
conjugate TCP with the gel of carboxymethylcellulose to
reestablish the articular cartilage. It was carried out an in
vitro evaluation of this biomaterial under sterile
conditions with growth factors and culture medium.
Several concentrations of this biomaterial were
encapsulated by cells of the matrix articulate starting
production of the new parent cartilage articular. The in
vitro results showed that the hydrogel presents great
potential for use in tissue engineering for repair articular
cartilage.
Keywords. Biomaterials, Composite, Articular Cartilage
(8.P4)
CHITOSAN-POLYVINYL
ALCOHOL
BASED
BIOPOLYMERS FOR AURICULAR NEOCARTILAGE USING
AUTOLOGOUS CELLS
Abarca E (1), Ruvalcaba E (1), Martínez V (1), García Z (2),
Luna-Barcenas G (2), Velasquillo C (1), Pérez M (1), Ibarra
C (1)
Medicine Unit, National Institute of Rehabilitation; 2.
Polymer & Biopolymer Research Group Cinvestav
Queretaro
Introduction. Reconstruction of cartilaginous structures
of the ear from autogenous tissues continues to be a
challenge in reconstructive surgery. In Mexico alone, 1 of
1500 children suffers from microtia (data from 1999) and
tissue engineering may provide insight for its treatment.
Biomaterials based on chitosan (CTS) and Poly (vinyl
alcohol) (PVA) show great potential for the creation of
synthetic cartilage. For all the above reasons the goals of
this study were (1) to engineer a biosynthetic construct
using CTS-PVA blends seeded with auricular cartilage, (2)
to study the feasibility of culture and proliferating
auricular cartilage in 3D while keeping normal cartilage
phenotype and (3) to compare the histology and
immunohistochemical composition of engineered
constructs.
Materials and methods. Pediatric auricular cartilage was
collected as excess tissue from ontological procedures
with parent consent. Cartilage was digested and cell
cultures were maintained in a monolayer culture.
Biopolymers of CTS-PVA were crosslinked with
epichlorohydrin (ECH) to cast films and were seeded with
auricular chondrocytes and cultured in standard in vitro
conditions. Cell viability onto the polymer was
determined by methylene blue assay and morphology
characteristics were studied by hematoxylin staining.
Environmental Scanning electron microscopy (ESEM) was
used to analyze cell adhesion to the polymer and
immunohistochemistry was performed to evaluate
production of cartilage proteins.
Results. Tissue’s histological evaluation tested positive to
proteoglycans, collagen and elastin. Statistic significance
was observed in cell viability and proliferation onto the
polymers when compared to a monolayer culture. Cells
had normal auricular morphological features and were
adhered to the polymer CTS-PVA-ECH analyzed by ESEM.
Immunohistochemistry showed constructs were positive
to collagen, elastin and aggrecan.
Conclusion. These results demonstrate the feasibility of
tissue-engineered cartilage as a potential graft material
for microtia treatment.
Acknowledgments. Partial financial support from grants
CONACYT 114359 and CONACYT 78798.
Keywords. auricular cartilage, tissue engineering,
Chitosan-Polyvinyl alcohol, constructs
(8.P5) THE EFFECTS OF AGAROSE ON CHONDROCYTE
DIFFERENTIATION IN A 3D CARTILAGE MODEL
Carriel V (1), Oliveira ACX (1), Garzón I (1), García JM (1),
Martín-Piedra MA (1), Moller A (2), Campos A (1)
1. Tissue Engineering Group, Dept. Histology, University of
Granada, Spain; 2. Biomedical Research Centre, School of
Medicine, Universidad de Valparaiso, Chile
Introduction.
The human
articular
cartilage is an
avascular conective tissue, which presents a low cell-to
matrix volume ratio with a highly specialized extracellular
matrix (ECM). Articular cartilage degeneration by
congenital abnormalities, disease and trauma could have
clinical consequences. Fibrin-Agarose (FA) biomaterial has
been previously used for the efficient generation of
cornea and skin substitutes. However, the influence of
this biomaterial on the biological behavior of human
hyaline chondrocytes and the ECM proteins that are
synthetized in culture are unknown. Here, we describe a
fibrin (F) and a fibrin-agarose 0.4% (FA) nanostructured
human cartilage substitute and evaluate the sequential
changes that take place in the ECM during five weeks of
culture.
Materials and methods. Human articular hyaline cartilage
biopsies obtained from healthy donors were
enzymatically digested with collagenase type II to
generate primary cultures of chondrocytes. Then, a
nanostructured human articular cartilage substitute was
developed in the laboratory using a fibrin and FA-0.4%
with condrocytes cultured within. Tissue samples were
analyzed after 1, 3 and 5 weeks of culture using
haematoxylin-eosin and alcian blue staining, and Ki-67
and laminin immunohistochemistry.
Results. The histological analysis revealed an increasing
number of cells with time in culture in both construct
types (F and FA). Alcian Blue staining was progressively
positive only in FA constructs, with higher signal after
longer times in culture. The inmunohistochemical analysis
for Ki-67 and Laminin was positive for both constructs
and for all weeks.
Conclusions. These results suggest that both fibrin and
fibrin-agarose biomaterials properly allow the progressive
prolifferation of the human hyaline chondrocytes
cultured within and the synthesis of laminin
glycoproteins. However, proteoglycans synthesis was
positive only when fibrin-agarose scaffolds were used. For
all these reasons, fibrin-agarose scaffolds are
recommended for the generation of an artificial human
hyaline cartilage.
Keywords.
nanostructure,
biomaterial,
cartilage,
proteoglycans, laminin
(8.P6) HUMAN CHONDROCYTES AND MESENCHIMAL
STEM CELLS RESPONSE TO A DECELLULARIZED HUMAN
DERMA
Fini M (1), Bondioli E (2), Melandri D (2), Giardino R (1),
Veronesi F (1), Giavaresi G (1)
1. Laboratory of Preclinical and Surgical Studies, Rizzoli
Orthopaedic Institute - IRCCS, Bologna – Italy; 2. Emilia
Romagna Skin Bank - Bufalini Hospital, Cesena – Italy
Introduction. Biological resurfacing has been advocated
as reconstructive treatment and several materials have
been proposed including extracellular matrix (ECM). The
aim of the study was to evaluate the biological response
of two human cell lines to a new decellularized human
dermal ECM membrane in comparison with a
commercially available human dermis.
Methods. Normal human articular chondrocytes (NHACkn) derived from human knee articular cartilage, and
human mesenchymal stem cells (hMSC) were seeded in
polystyrene wells (TCP) as controls, and on a
decellularized human dermis from multi-organ donors
(HDM_derm) and GRAFTJACKET® – Maximum Force
membrane (GJ) for 7 and 14 days.
Results. NHAC-kn and hMSC proliferation was higher on
HDM_derm than it was on GJ at both experimental times.
Phenotype expression was maintained on both tested
membranes, for NHAC-kn, while hMSC cultures showed
significant increases in pro-cathepsin B (108%, p < 0.005)
and CPII (12%, p < 0.05). The synthesis of TGF-β1 was
higher in hMSC where significantly higher values were
found when cultured on GJ than HDM_derm at both 7
(152%, p <0.0005) and 14 (43%, p <0.005) days with
significant increases between the two experimental time
for cultures seeded on GJ (237%, p < 0.005) and
HDM_derm (92%, p < 0.0005).
Conclusions. The results obtained showed that
HDM_derm seems more suitable than GJ for the
differentiation and growth of the NHAC-kn. Further
investigations are mandatory to understand better the
behaviour of hMSC, above all for their expression towards
a chondrogenic phenotype when in contact with
HDM_derm. This study represents the first evidence to
support the use of a HDM_derm with this new method as
a scaffold for soft tissue regeneration with special interest
for biological resurfacing.
Keywords. Decellularized human dermal matrix,
chondrocytes, mesenchimal stem cells
(8.P7) GLOBAL GENE EXPRESSION ANALYSIS OF
MESENCHYMAL
STROMAL
CELLS
FROM
OSTEOARTHRITIC DONORS
Rauh J (1), Friedrich H (1), Overall R (2), Royer L (3),
Kruhoffer M (4), Günther K-P (1), Stiehler M (1)
1. Department of Orthopedics and Centre for
Translational Bone, Joint and Soft Tissue Research,
University Hospital Carl Gustav Carus, Dresden, Germany;
2. Center for Regenerative Therapies Dresden, Dresden
University of Technology, Dresden, Germany; 3.
Biotechnology Centre of the University of Technology
Dresden, Dresden, Germany; 4. Molecular Diagnostic
Laboratory, Clinical Chemical Department, Aarhus
University Hospital, Denmark
Osteoarthritis (OA) is one of the most frequent
musculoskeletal disorders and represents the main
indication for total joint arthroplasty. However, the exact
aetiology of OA remains the focus of ongoing research.
Mesenchymal stromal cells (MSCs) can be easily isolated
from bone marrow aspirates and provide an excellent
source of progenitor cells. Previously differences in
proliferation and differentiation of MSCs from
osteoarthritic versus healthy donors were reported. To
elucidate the role of MSCs in OA aetiology we compared
global gene expression of MSCs derived from
osteoarthritic versus healthy donors.
MSCs were isolated from bone marrow aspirates of n=13
advanced-stage osteoarthritic and n=15 age-matched
healthy donors by density gradient-centrifugation and
polystyrene adhesion. After cell expansion until
subconfluency total RNA of MSCs at passage 0 were
analysed using Affymetrix® GeneChip Human Genome
U133 Plus 2.0 Arrays. Raw data were processed by
background correction, normalization, and robust
multichip analysis followed by statistical analysis using
“R” and one-way ANOVA for gender-related or intergroup
gene expression differences. Gene ontology (GO) and
pathway analyses were performed by use of NetAffx™,
DAVID, KEGG, and Babelomics4.
A total of n=690 intergroup differentially regulated genes
were identified. Notably the most significantly regulated
gene, component of oligomeric golgi complex 5, had
recently been reported to be associated with an
increased risk of OA. Using GO the functions of genes
differentially regulated in OA-MSCs were classified into
processes
of
transport,
transcription,
protein
modification, apoptosis, RNA modification, and cell
adhesion. Notch, Wnt and Jak-Stat were identified as the
most significantly affected signal transduction pathways
by OA in MSCs.
Using global gene expression analysis of MSCs from
osteoarthritic versus healthy donors we identified
relevant candidate genes and signal transduction
pathways. These data support the hypothesis that MSCs
play a central role in the aetiology of osteoarthritis and
warrant further studies.
Keywords. osteoarthritis, mesenchymal stromal cells,
gene expression analysis, microarray
(8.P8)
INTEGRATING
BIOMIMETIC
BIOREACTOR
CONDITIONS AND ALGINATE MICROBEADS TO INDUCE
FORMATION OF CARTILAGINOUS TISSUE CONSTRUCTS
Obradovic B (1), Stojkovska J (1), Kostic D (1)
1. Faculty of Technology and Metallurgy, University of
Belgrade
Particulate cell supports, especially in the form of
microbeads, provide short diffusion distances for efficient
mass transfer to cells, as well as possibilities for minimally
invasive implantation by injection and environments for
uniform cell distribution and extracellular matrix (ECM)
regeneration. In addition, interstitial channels create
structures enabling development of vasculature between
individual particles. We have previously shown that
alginate microbeads were suitable supports for
immobilization of chondrogenic cells and cartilaginous
ECM regeneration in perfusion bioreactors. Bonding of
the beads and formation of continuous cartilaginous
constructs depended on the cell density, bead size, and
medium flowrate. However, since articular cartilage is
normally exposed to dynamic loads, in this study we have
utilized a novel biomimetic bioreactor integrating
dynamic compression together with tissue perfusion
applied in physiological regimes in order to enhance
regeneration of cartilaginous tissue. Packed beds of
alginate microbeads (~900 µm in diameter) with
immobilized bovine calf chondrocytes (33x106 cells/ml)
were cultivated over 4–6 weeks under dynamic
compression (1h on/1h off, frequency 0.4 - 0.6Hz, 10%
strain) and medium perfusion (flowrate of 0.28 ml/min
corresponding to the superficial velocity of 25µm/s). The
bioreactor provided also monitoring of biomechanical
properties of the packed beds over the cultivation time
(Fig. 1). Compression moduli decreased in the first two
weeks of cultivation due to alginate gel weakening but
then started to increase as the cells produced ECM. After
4 weeks of cultivation large bonded groups of microbeads
were formed. Results of this study can be relevant not
only for cartilage tissue engineering but also for
controlled studies of particulate cell supports under
conditions that imitate physiological in vivo environments
as well as for predictions of implant behavior.
Keywords. biomimetics, bioreactor, cartilage tissue
engineering, dynamic compression, alginate
Figure 1. Compression modulus of packed beds of
microbeads over 4 weeks of cultivation; insert: merged
beads after 4 weeks of cultivation
(8.P9) MIGRATION OF CHONDROCYTES FROM ADULT
HUMAN CARTILAGE INTO BIOMIMETIC SCAFFOLDS
Wardale J (1), Hopper N (1), Ghose S (2), Rushton N (1)
1. Univesity of Cambridge, UK; 2. Tigenix, UK
Introduction. Biomimetic scaffolds hold great promise for
therapeutic repair of cartilage and bone but still require
optimization in terms of their ability to integrate with the
host tissue in order to establish an appropriate
extracellular matrix (ECM). Adult human cartilage has a
limited capacity for repair but there is evidence that
chondrocytes can migrate and maintain some
chondrogenic phenotype (1). This study investigated the
conditions under which chondrocytes migrate out of
cartilage and into biomimetic scaffolds in vitro.
Materials and Methods. Articular cartilage explants were
cut from femoral condyles and tibial plateux derived from
donors undergoing total knee replacement for
osteoarthritis (OA). Cultures were maintained for up to 28
days and examined by light microscopy and
immunohistochemistry. Explants were also studied using
an xCelligence apparatus (Roche) to measure real time
cell migration.
Results. Cells migrated to the periphery of cartilage
explants after approximately 10 days in culture where
they proliferated and deposited an extracellular matrix.
The number of migratory cells was related to the location
of the original cartilage, cutting method, culture
conditions and could be manipulated by the addition of
growth factors. Migration of cells and deposition of an
ECM into a collagen/glycosaminoglycan scaffold was
considerably enhanced by the addition of growth factors.
Discussion. The precise interaction between biomimetic
scaffolds and damaged host tissue is seldom investigated
prior to in vivo studies. It may be possible to exploit the
ability of human OA cartilage as a source of migratory
cells to aid the population of biomimetic scaffolds thus
enhancing the repair process. Studies are currently in
progress to identify and manipulate the phenotype of this
migratory cell population.
References. Morales, T. Osteoarthritis Cartilage. 2007
15(8): 861–871
Keywords. Cartilage Repair, Biomimetic Scaffold,
Chondrocyte, Migration
(8.P10) COMPARISON OF PLACENTAL AND BONE
MARROW-DERIVED STEM CELLS FOR CARTILAGE TISSUE
ENGINEERING.
Crawford A (1), Frias AM (2), Hatton PV (1), Reis RL (2),
Redl H (3), Hildner F (3)
1. University of Sheffield; 2. University of Minho; 3. Ludwig
Boltzmann Institute for Experimental & Clinical
Traumatology
Introduction. Stem cells offer great potential for
regenerative medicine technologies. Embryonic stem
cells, in contrast to adult mesenchymal stem cells (MSCs),
exhibit pluripotency and can differentiate into many
different cell types. A less invasive source of potential
foetal-derived stem cells, are the amniotic fluid and
placenta.
AIM. The aim was compare human amnion (HAM),
amniotic fluid (HAF) and bone marrow-derived MSCs
(BM-MSCs) for their chondrogenic potential for cartilage
tissue engineering.
Methods. HAM, HAF and BM-MSCs were seeded
dynamically onto PGA scaffolds after the expansion of the
cell numbers in monolayer culture. The resultant
constructs were cultured for 4 weeks in classical
chondrogenic media (DMEM containing 1mg/ml BSA,
insulin/transferrin/selenium, 10-7M dexamethasone,
25µM ascorbic acid, TGFß). At 2 and 4 weeks, constructs
were taken for analysis of gene expression by real-time
PCR (AGC1, COL2A1, COL9A2, COL10A, SOX9, MIA, CRTL1,
CSPG2 and COMP). After 4 weeks, proteoglycans
(detected as glycosaminoglycans (GAG) were measured
using dimethylmethylene blue and frozen sections taken
for histochemical and immunochemical analysis of the
extracellular matrix.
Results. BM-MSCs accumulated the most extracellular
matrix containing collagen II and proteoglycan. BM-MSCs
showed the highest expression of markers for hyaline
cartilage (AGC1, COL2A1, COL9A2, SOX9, CTLR1, MIA) and
accumulation of GAG. Expression of collagen I was similar
for BM-MSCs and HAF cells and lower in HAMs. BM-MSCs
also showed the highest expression of the hypertrophic
marker, collagen X. Neither collagens II, X nor aggrecan
was expressed by HAM cells.
Conclusions. Under the conditions used, BM-MSCs were
the more appropriate stem cell type for cartilage tissue
engineering but displayed a tendency to hypertrophy.
Acknowledgements. This collaborative research would
not have been possible without funding from the
European
Commission
which
established
the
EXPERTISSUES Network of Excellence [reference 500283].
A.M. Frias is recipient of a post-doctoral scholarship from
Fundação
para
a
Ciência
e
Tecnologia
(SFRH/BPD/45206/2008).
Keywords. stem cells, placental, bone marrow, cartilage
tissue engineering
(8.P11) MORPHOLOGICAL AND MOLECULAR ANALYSIS
OF THE INTERACTIONS AMONG BONE, CARTILAGE AND
BIOMATERIALS BY MICROSCOPY NMR
Esteve V (1), Martín de Llano J (1), Martínez-Bisbal MC
(1), Sancho-Tello M (1), Celda B (1), Carda C (1)
1. Departamento de Química Física, Universitat de
Valencia, Valencia Spain; CIBER-BBN, Universitat de
Valencia, Valencia, Spain; Universidad de Valencia,
Facultad de Medicina, Departamento de Patología.
INCLIVA; CIBER-BBN
Introduction: The deeper understanding of detailed
interactions among bone, cartilage and biomaterials will
allow obtaining essential information for improving the
design of most biological compatible materials.
Microscopy Nuclear Magnetic Resonance (MNMR) is a
non-destructive
technique
that
can
provide
morphological description of the biological sample and, at
high
magnetic
fields,
molecular
information.
Morphological and molecular initial results in fresh and
fixed rabbit’s knee bone samples by NMRM will be
discussed. A preliminary NMRM approach for a
biomaterial/bone knee sample will be presented.
Methods: New Zealand rabbit’s knee samples, fresh or
fixed and decalcified have been studied by MNMR at 14T.
Different MRI microscopy pulse sequences and distinct
MR parameters have been tested. MNMR morphological
2D and 3D images by using MSME, MDEFT, FLASH and MMSME sequences with different in plane and section
resolution have been obtained. Likewise, PRESS single
voxel spectra at short echo time have been acquired.
SE3D, GE3D and SPI3D MNMR images have been acquired
for a pilot biomaterial/bone knee sample included in
PMM.
Results: An example of morphological images by MNMR
in fresh and fixed rabbit’s knee is shown. Particularly
significant is the adequate resolution between bone and
cartilage achieved by MDEFT technique. M_MSME images
show important constitutive regions of the bone part. In
addition, differences can be observed in the cartilage and
bone structures between both samples. The metabolic
profiles of the fresh sample are different at distinct
regions. Different parts of the bone and cartilage can be
observed in the biomaterial/bone knee sample by MNMR
images.
Conclusions: MNMR can provide morphological and
molecular information complementary to histology and
CT. Some structural differences have been observed
between fresh and fixed samples by MNMR. MNMR, as
non-invasive technique, can be applied in nonmanipulated biological samples and its results can be
translated to MRI in vivo applications.
Figure 1. NMR microscopy images (MDEFT and M_MSME)
for two different knee’s rabbit samples: - left part: fresh
sample; - right part: decalcified and fixed sample.
(8.P12) LOW OXYGEN TENSION IS NOT BENEFICIAL FOR
THE NEOCARTILAGE FORMATION IN SCAFFOLD-FREE
PRIMARY CHONDROCYTE CULTURES
Qu C (1), Lindeberg H (1), Lammi MJ (1)
1. Department of Biosciences and Biocenter Kuopio,
University of Eastern Finland, Kuopio, Finland
Introduction. Articular cartilage is an avascular tissue that
lives at low oxygen (O2) environment in vivo. The present
study was aimed to investigate whether 5% O2 could be
beneficial for the neocartilage formation when bovine
primary chondrocytes were cultured in type II collagencoated membrane insert, and whether hyaluronan (HA)
or glucosamine sulfate (GS) would enhance the
extracellular matrix production of the neocartilage when
the cells are cultured in insert at 5% oxygen tension.
Methods. Primary chondrocytes isolated from articular
cartilage of bovine femoral condyles were seeded into
insert at the cell density of 6 million, and cultured in
DMEM supplemented with 10% FBS and antibiotics
(control), or GS or HA at 5% or 20% O2 atmosphere for 2,
4, and 6 weeks. The samples were then collected for
histological staining of PGs and type II collagen, qRT-PCR
of aggrecan and procollagen α1(II) mRNA expressions,
and PG content quantification.
Results. Neocartilage produced at 20% O2 appeared
larger than at 5% O2, and it was larger and more
homogenous in GS-treated cultures than in other cultures
at 20% O2. Histological staining showed that more PGs,
type II collagen and better native cartilage structure was
produced at 20% compared to 5% O2. The thickness of
neocartilage increased following culture period.
Quantitative RT-PCR showed that aggrecan and
procollagen α1(II) mRNA expressions were significantly
higher at 20% O2, as well as PG content. However, no
significant difference in gene expression and PG content
found between control and GS- or HA-treated culture
either at 20% or 5% O2.
Conclusions. We conclude that, in contrast to monolayer
cultures, engineered-cartilage from scaffold-free primary
chondrocytes at 20% O2 produced better extracellular
matrix production than at 5% O2. The PGs mainly
consisted of large ones. However, exogenous GS or HA
was not beneficial for the ECM production in scaffold-free
cultures.
Keywords. 5% oxygen, scaffold-free, neocartilage
(8.P13) METHOD FOR SCAFFOLDING TO CARTILAGE
TISSUE SUBSTITUTION A TRACHEAL APPROACH
Giraldo D (1), Piña M (1), Villegas F (2)
1. Instituto de Investigaciones en Materiales-Universidad
Nacional Autonoma de Mexico; 2. Departamento de
Cirugia-Facultad de Medicina-Universidad Nacional
Autonoma de Mexico
The present paper present the early results about of
different methods for obtain a xenograft for a tracheal
tissue cartilage substitution, was used four method to
wash the trachea segment from porcine using a
enzymatic detergent, and partial enzyme degradation to
remove cell material own of the extracellular matrix and
avoid the immune reaction, was done the
characterization by Scanning Electron Microscopy (SEM)
and histology to evaluate the cell removing and
morphological change in the extracellular matrix, the
samples were further characterized by thermal
techniques like Thermo Gravimetric Analysis (TGA) and
Differential Scanning Calorimetric (DSC), furthermore the
cell viability was measured by cell culture and the
biological response were evaluated by implantation in
New Zealand Rabbits. The first results shown that the
treatment with enzyme degradation is the most effective
to remove the cellular material and avoid the immune
reaction.
Keywords. Cartilage, Scaffold, Trachea
(8.P14) EVALUATION OF HYALURONIC ACID-CHITOSAN
SPONGES FOR CARTILAGE TISSUE ENGINEERING: INVITRO AND IN-VIVO STUDIES
Demirdögen B (1), Elcin AE (2), Elcin YM (1)
Science, TEBNL, Ankara, Turkey; 2. Ankara University,
Stem Cell Institute, Faculty of Science, TEBNL, Ankara,
Turkey; Gazi Univ., GEF, Biology Div., Ankara, Turkey
Introduction. The aim of this study was to develop a
scaffold system based on hyaluronic acid (HA) and
chitosan (C) that could support the proliferation and
chondrogenic
differentiation
of
bone
marrow
mesenchymal cells (BM-MSCs) for cartilage tissue
engineering.
Methods. HA-C sponges were prepared by forming a
polyelectrolyte complex precipitate from aqueous blends
of HA and C. The final product was attained after
lyophilization and crosslinking. SEM and FTIR were used
to evaluate morphology and chemical composition,
respectively. The swelling ratio and in-vitro degradation
rate of the sponge were determined. In-vitro cell culture
experiments were performed using rat BM-MSCs and rat
articular chondrocytes (ACs; as control), under
chondrogenic conditions (+TGF-β1). Cell distribution and
morphology were followed. Cell viability and proliferation
were determined by the MTT assay; total GAGs synthesis
were determined as well. Cellular sponges were
subcutaneously transplanted into Wistar rats and
explanted cellular grafts were evaluated by histology.
Results. SEM demonstrated that HA-C sponges had a
highly porous structure composed of interconnecting
pores providing a suitable environment for cell migration.
FTIR spectra of the HA-C sponge showed the expected
peaks of HA and C indicating the presence of both
polymers within the sponge. HA-C sponge was
mechanically stable under the culture conditions for the
duration of in-vitro studies. The seeded BM-MSCs and
ACs adhered and proliferated inside sponges as indicated
by the MTT test. Total sGAG secreted by the
differentiated BM-MSCs were comparable with that of
ACs. Chondrogenic differentiation of BM-MSCs was
confirmed by histology. In-vivo studies revealed that both
cellular and acellular sponges showed a mild level of
tissue reaction. Histology also confirmed the homogenous
distribution of the cells and cartilage-like tissue formation
inside the HA-C sponges.
Conclusions. These data seem to indicate that HA-C
sponges provide a suitable 3D-scaffold environment for
cartilage tissue engineering.
Keywords. hyaluronic acid, chitosan, mesenchymal stem
cells, chondrogenesis
(8.P15) PLATELET-RICH PLASMA HAS ANTI-CATABOLIC
PROPERTIES IN OSTEOARTHRITIC CHONDROCYTES
van Buul GM (1,2), Koevoet WLM (3), Kops N (1), Bos PK
(1), Verhaar JAN (1), Weinans H (1,4), Bernsen MR (2), van
Osch GJVM (1,3)
1. Department of Orthopaedics, Erasmus MC, Rotterdam,
the Netherlands; 2. Department of Radiology, Erasmus
MC, Rotterdam, the Netherlands; 3. Department of
Otorhinolaryngology, Erasmus MC, Rotterdam, the
Netherlands;
4.
Department of
Biomechanical
Engineering, Delft University of Technology, Delft, the
Netherlands.
Introduction. Platelet-rich plasma (PRP) has recently been
postulated as a treatment for osteoarthritis (OA).
Although anabolic effects of PRP on chondrocytes are
well documented, no reports are known addressing anticatabolic responses. Since OA is characterized by a
catabolic joint environment, we studied whether PRP
exerted anti-catabolic effects on primary human
osteoarthritic chondrocytes.
Methods. PRP was prepared from whole blood from
three healthy donors. Human OA chondrocytes from six
donors were cultured in alginate beads in the presence of
IL-1β to mimic an osteoarthritic environment. Medium
was supplemented with 0%, 1% or 10% PRP releasate
([PRPr] the active releasate of PRP). After 48 hours, gene
expression of collagen type II (COL2), aggrecan (AGCN), a
disintegrin and metalloproteinase with thrombospondin
motifs (ADAMTS)-4, ADAMTS-5, matrix metalloproteinase
(MMP)-13 and cyclo-oxygenase (COX)-2 was analyzed.
Additionally, glycosaminoglycan (GAG) content, nitric
oxide (NO) production and nuclear factor kappa B (NF-κB)
activation were studied.
Results. IL-1β diminished expression of the anabolic
genes COL2 and AGCN in chondrocytes, while it increased
expression of the catabolic ADAMTS-4, MMP-13 and COX2 (P<0.03 for all genes). PRPr diminished IL-1β induced
inhibition of COL2 (P=0.003) and AGCN (P=0.001) gene
expression. PRPr also reduced IL-1β induced increase of
ADAMTS-4 (P=0.001) and COX-2 (P=0.004) gene
expression. ADAMTS-5 gene expression and GAG content
were not influenced by IL1-b nor PRPr. MMP13 gene
expression and NO production were upregulated by IL-1β
but not affected by PRPr. Finally, PRPr reduced IL-1β
induced NF-κB activation to control levels containing no
IL-1β (P<0.001).
Conclusions. PRPr diminished multiple catabolic IL-1β
effects in human osteoarthritic chondrocytes. PRPr
exerted anti-catabolic effects on genes regulating
extracellular matrix formation, as well as inflammation, in
human chondrocytes. Moreover, PRPr decreased NF-κB
activation, a major pathway involved in the pathogenesis
of OA. These results encourage further use and study of
PRP as a treatment for OA.
Keywords. platelet-rich plasma; PRP; cartilage;
chondrocyte; osteoarthritis; anti-catabolic
(8.P16)
CO-CULTURE
OF
MONOCYTES
AND
MESENCHYMAL STEM CELLS UNDER HYPOXIC
CONDITIONS
Hopper N (1), Wardale J (1), Ghose S (2), Rushton N (1)
1. Orthopaedic Research Unit, University of Cambridge,
Box 180, Addenbrooke's Hospital, Hills Road, Cambridge
CB2 0QQ, United Kingdom; 2. Tigenix, Byron House,
Cambridge Business Park, Milton Road, Cambridge, CB4
0WZ, United Kingdom
Introduction. The overarching goal of therapeutic
cartilage repair is to adopt a biomaterial to deliver cells
and growth factors into the articular cartilage defect to
facilitate the healing response. Adult cartilage is an
avascular tissue; thus an injury is not followed by an influx
of monocytes. Mesenchymal stem cells (MSCs) stand out
as an ideal progenitor cell source for cartilage tissue
engineering. This study describes the development of
osteochondral graft consisting of primary monocytes and
MSCs on a collagen biomaterial scaffold.
Materials and Methods. A novel collagen-GAG scaffold
(Chondromimetic®, Tigenix) composed of type I collagen
and
chondroitin
sulphate
provided
a
3D
microenvironment for the osteochondral graft. Ovine
MSCs were a gift from Mesoblast (Australia) and primary
ovine monocytes were prepared by centrifugation of
fresh, heparinized whole blood using Lymphoprep™.
MSCs were plated either in the scaffold or as
micromasses on tissue culture plastic. After 24 hours
monocytes were added in various concentrations.
Cultures were maintained for up to 21 days both in
normoxic and hypoxic cell culture conditions.
Results. Ovine MSCs maintained their characteristics in
the presence of monocytes. After 2 weeks in culture, the
extra cellular matrix production (ECM) was increased by
the addition of monocytes. Hypoxic culture conditions
were found to have a positive effect on the monocyte
activity. The collagen-GAG biomaterial scaffold supported
the cell attachment and proliferation towards
chondrogenic lineage.
Discussion. The molecular interaction between
monocytes and MSCs has yet to be established but is has
been thought to involve paracrine factors. The results of
this study suggest that each cell type, monocyte and MSC,
may contribute independently of each other in supporting
the osteochondral graft properties. Further studies are
underway to evaluate whether the positive effects of
monocytes are due to an adherent progenitor cell
population in the circulating monocytes.
Keywords. cartilage repair, MSC, hypoxia
(8.P17) IN VITRO AND IN VIVO EVALUATION OF
HA/CARBON FABRICS SCAFFOLD FOR CARTILAGE REPAIR
Rajzer I (1), Menaszek E (2), Bacakova L (3), Blazewicz M
(4)
1. Institute of Textile Engineering and Polymer Materials,
ATH University of Bielsko-Biala, Poland; 2. Departament
of Cytobiology, Collegium Medicum, UJ Jagiellonian
University, Poland; 3. Institute of Physiology, Academy of
Sciences of the Czech Republic, Czech Republic; 4.
Department of Biomaterials, Faculty of Materials Science
and Ceramics, AGH University of Science and Technology,
Poland
Introduction. Carbon fibers have been widely
investigated as cellular growth supports in cartilage tissue
engineering. However, long duration of the process of
cartilage restoration limits the applicability of CFs
implants in the treatment of cartilage tissue defects.
Hyaluronic acid plays a key role in cartilage tissue
development, repair and function. In the present study
we focused on the in vitro and in vivo evaluation of two
types of carbon nonwoven fabrics: hyaluronan modified,
and non-modified carbon nonwovens.
Experimental Methods. Hyaluronic acid sodium salt (HA)
was purchased from CPN spol s.r.o. (Czech Republic).
Carbon nonwoven fabrics (CNFs) were prepared from
polyacrylonitrile precursor via two-stage process:
stabilization (150-280°C) and carbonization (1000°C,
argon). Hyaluronic acid (HA) was immobilized onto the
surface of macroporous carbon nonwoven fabrics. The
cytotoxicity of the samples (CNF and CNF_HA) were
determined in the culture of human lung adenocarcinoma
cell line A549. MG-63 cells (European Collection of Cell
Cultures, Salisbury, UK) were used for testing the cellmaterial interaction in vitro. The knee cartilage of rabbits
was used as a model tissue for in vivo studies.
Results And Discussion. Direct contact of the cell line
A549 did not show any cytotoxicity effect neither in CNF
nor in CNF_HA. Adhesion of MG-63 cells was better on
CNF_HA composites than on unmodified carbon fabrics.
MG-63 cells adhered well to carbon fibers showing an
elongated shape. Knee defects treated with CNF_HA were
repaired in different degree with hyaline-like cartilage
tissue, granulation tissue and bone. Numerous capillaries
present in regenerating tissue allow to expect the proper
reconstruction of chondral and bone tissues.
Conclusion. Modified carbon nonwovens used in our
study are a promising scaffold which allows cells to grow
within it and to form new cartilage and bone.
Acknowledgments. Work supported by Polish Ministry of
Science and Higher Education (Iuventus Plus:
IP2010034270).
Keywords. scaffolds, fibers, cartilage, hyaluronic acid
(8.P18) DESIGN OF EXPERIMENTS AND RESPONSE
SURFACE MODELLING FOR OPTIMISATION OF DEFINED
CHONDROGENIC MEDIUM
Enochson L (1), Lindahl A (1)
1. Biomedicine
Introduction. The golden standard of defined media for
cartilage differentiation in cartilage tissue engineering
was originally optimised for Mesenchymal Stem Cells
(MSCs) 12 years ago by Johnstone et al[1]. It was
thereafter applied to human chondrocytes by Tallheden
et al [2] and others. The medium has, however, never
been optimised for chondrocytes. In this abstract we
show the usefulness of computer based design of
experiments (DoE) and response surface modelling (RSM)
for optimisation of a defined medium for tissue
engineering of articular cartilage with human
chondrocytes.
Methods. Surplus chondrocytes from two patients (mean
age 18±2 yrs) undergoing autologous chondrocyte
implantation (ACI) were expanded in DMEM/F12 and 10%
human serum. The cells we cultured for two weeks in
pellet mass culture in 17 different medium formulations
designed in Modde 8.0 (Umetrics AB, Sweden), with 6
variables (TGFb1, Dexamethasone, Human serum
albumin, ITS+, Ascorbic acid, Glucose). The pellet size was
assessed and RNA was extracted after two weeks of
culture. Expression of matrix components were assessed
with qPCR. The results were displayed in response
surfaces (fig 1).
Results. TGFbeta1, dexamethasone and glucose showed
to be significant factors for pellet size and matrix
components expression. Optimized medium for
chondrocyte differentiation was 13 ng/ml TGFbeta1
(+30%), 50 nM dexamethasone (-50%) and 5.5 g/L glucose
(+22%), compared to the golden standard (p<0.009).
Conclusion. The golden standard culture media in
cartilage tissue engineering needs to be revised for
optimal culture processes. Computer based DoE and RSM
are powerful tools for this and other culture optimization
purposes.
References
1. Johnstone et al, Exp Cell Res. 1998;238(1):265-72 l
2.
Tallheden
et
al,
Osteoarthritis
Cartilage.
2004;12(7):525-35.
Keywords. Cartilage, Design of Experiments, Response
Surface Modelling, Medium Optimisation
(8.P19) HUMAN OSTEOARTICULAR COMPLEXES:
CHARACTERIZATION AND EVOLUTION OF PRESEEDED
CHONDROCYTE BIOMATERIALS/SCAFFOLDS USING A
NUDE MICE MODEL
Martín de Llano JJ (1), Sancho-Tello M (1), Gamboa T (2),
García Cruz D (3), Forriol F (4), Gastaldi P (4), GómezRibelles JL (2,3), Carda C (1)
1. Departamento de Patología, Facultad de Medicina y
Odontología, Universidad de Valencia; INCLIVA; CIBER
BBN; Valencia, Spain; 2. Center for Biomaterials and
Tissue Engineering, Universidad Politécnica de Valencia,
Spain; 3. Centro de Investigación Príncipe Felipe,
Regenerative Medicine, Valencia, Spain; 4. Hospital de la
Malvarrosa, Valencia, Spain
Introduction. The aim of the present work is to study the
response of human chondrocytes seeded in a 3D
construct consisting in biomaterial microspheres
embedded in a fibrin clot, a model that provides the cells
with a human osteochondral environment while
implanted in nude mice.
Methods. Osteochondral cylinders (1-cm diameter) were
obtained from healthy areas of the tibial plateau of
patients undergoing knee replacement surgery. A
centered 3-mm cavity was drilled on the articular surface,
which was filled with microspheres of a biomaterial
preseeded with human chondrocytes, and mixed with
fibrin. Three polymers were tested: chitosan with or
without hialuronic acid and polycaprolactone. The
cylinders were subcutaneously implanted on the back of
nude mice. Animals were sacrificed after 1, 2 and 4 weeks
of surgery, and cylinders were removed and processed for
histological and immunohistochemical (type-I and -II
collagens, and human nuclear-antigen detection) analysis.
Results. Most of the microspheres remained in the cavity
4 weeks after implantation of the osteochondral cylinder.
Histological analysis showed an inflammatory response,
assessed by neutrophil infiltration, when chitosan or
chitosan-hyaluronic acid were used as biomaterials. No
signs of inflammation were observed when
polycaprolactone was used; furthermore, Masson’s
trichrome stain showed the deposition of new cartilagelike extracellular matrix in these samples, which was
confirmed by immunodetection of type-II collagen.
Conclusions. From the three polymers tested, fibrin-glued
polycaprolactone
spheres
showed
a
better
biocompatibility. This biomaterial favored the synthesis of
cartilage-like extracellular matrix. The human or murine
origin of the cells responsible of the new extracellular
matrix is under study. These results indicate the feasibility
of polycaprolactone spheres injection for the restoration
of the articular cartilage.
Acknowledgements. Parly supported by Spanish MCINN
grants MAT2007-66759-C03-01-03 and MAT2010-21611C03-01-03.
Keywords. Human osteochondral complex; chondrocyte;
scaffold
(8.P20) HYDROSTATIC PRESSURE AND TGF-β3 INTERACT
TO REGULATE CHONDROGENESIS OF JOINT TISSUE
DERIVED MESENCHYMAL STEM CELLS
Vinardell T (1), Buckley CT (1), Meyer EG (1), Kelly DJ (1)
1. Trinity College Dublin
Introduction. Hydrostatic pressure (HP) is a key
component of the joint mechanical environment and has
been shown to enhance chondrogenesis of chondrocytes
(CC) and mesenchymal stem cells (MSCs). The objective of
this study was to investigate the interaction between HP
and TGF-β3 in regulating chondrogenic differentiation of
joint derived MSCs.
Methods. MSCs were harvested from synovial membrane
(SM) and infrapatellar fat pad (FP) of the femoro-patellar
joint, centrifuged to form pellets and subjected to 10 MPa
of HP at 1Hz for 4 hours (14 days), controls were
maintained in free swelling (FS) conditions. Pellets were
cultured with different concentrations of recombinant
human TGF-β3 (0, 1 and 10 ng/ml). Samples were
analysed biochemically (Glycosaminoglycan (GAG) and
collagen content) and histologically.
Results. SM and FP derived MSCs underwent robust
chondrogenesis in FS conditions when supplemented with
10 ng/ml TGF-β3, HP having no effect at this TGF-β3
concentration. In contrast at 1 ng/ml TGF-β3, HP
significantly increased GAG accumulation. The same trend
was observed for collagen accumulation. Chondrogenesis
was not observed in the pellets cultured in the absence of
TGF-β3 in either the FS or HP groups. HP appeared to
influence the organization of the neo-tissue, as evidenced
with the appearance of a core region in the FS pellets
compared to more homogeneous organization in the HP
group (Fig.1).
Conclusion. HP alone did not induce chondrogenesis, but
when applied with low concentration of TGF-β3, it acted
to promote chondrogenesis for FP and SM MSCs. At high
magnitudes of TGF-β3, HP had no additional synergistic
effect on chondrogenesis, suggesting an upper limit on
the stimulatory effects these two chondrogenic cues can
have. Future studies will investigate the influence of HP
on the development and organization of cartilaginous
tissues engineered using joint tissue derived MSCs.
Acknowledgements. Supported by Science Foundation
Ireland [SFI/08/YI5/B1336]
Keywords. MSC, chondrogenesis, hydrostatic pressure,
TGF-β3
(8.P21) POTENTIAL GENE SPECIFIC MARKERS TO PROOF
THE PURITY OF HUMAN ARTICULAR CHONDROCYTES IN
MONOLAYER CULTURE
Van der Lee J (1), Hagman M (1), Brantsing C (1), Brittberg
M (2), Dehne T (3), Ringe J (3), Lindahl A (1)
1. Department of Clinical Chemistry and Transfusion
Medicine, Sahlgrenska University Hospital, Gothenburg,
Sweden; 2. Cartilage Research Unit, University of
Gothenburg, Department of Orthopaedics, Kungsbacka
Hospital, Kungsbacka, Sweden; 3. Tissue Engineering
Laboratory and Berlin-Brandenburg Center for
Regenerative Therapies, Department of Rheumatology
and Clinical Immunology, Charité-U
Introduction. Due to the new EC regulations, No
1394/2007 on Advanced Therapy Medical Products, there
is a need to establish markers to proof the purity of
human articular chondrocytes in monolayer culture
before treatment. The most likely contaminants in the
cultures are synoviocytes due to synovial overgrowth of
the biopsy area. The aim of the study was to (i) study
chondrogenic differentiation of synoviocytes and to (ii)
identify genes that could specifically determine purity
regarding synoviocyte contamination.
Methods. Synoviocytes were isolated from human tissues
(n=5) and the cells were expanded in monolayer culture
(ML) followed by RNA preparation or seeding to a
hyaluronan scaffold (HYAFF 11, Fidia Advanced Polymers)
subsequently cultured for 14 days in a modified
differentiation media. The scaffolds were analyzed
regarding handling property, morphology and histology.
Messenger RNA from ML cultures was subjected to gene
expression analysis using oligonucleotide microarray
(Affymetrix). Expression data was compared to previous
microarray data from human chondrocytes in ML (n=5).
Candidate genes selected from the microarray analysis
were confirmed by real-time PCR.
Results. When comparing monolayer chondrocytes with
synoviocytes
no
differences
were
observed
microscopically and synoviocyte seeded scaffolds showed
similar handling characteristics as chondrocyte seeded
scaffolds. However, the histology results showed slightly
higher matrix production in the chondrocyte seeded
scaffolds. The gene expression comparison identified a
distinct set of 4 genes (designated Syn 1- 4) that were
barely detected in chondrocytes but highly expressed in
synoviocytes.
Conclusions. Although the articular cartilage and the
synovium tissue can be easily identified in biopsies, it is
impossible to exclude potential contaminating
synoviocytes in the subsequent culture process. We here
demonstrate that three of the identified candidate genes
have the potential for identifying chondrocyte purity
regarding synoviocyte contamination.
Keywords. Purity, gene marker, chondrocytes,
synoviocytes
(8.P22) MICROENCAPSULATION OF CHONDROCYTES IN
THE ALGINATE –CHITOSAN-ALGINATE SYSYTEM
Wasiak I (1), Butruk B (1), Ciach T (1)
1. Warsaw University of TechnologyFaculty of Chemical
and Process Engineering
Introduction. Microencapsulations of chondrocytes in the
alginate-chitosan-alginate (ACA) microcapsules, fibers and
sheets were performed. Both chitosan and alginate have
chemical composition and properties similar to the
cartilage, what makes them the material of choice for
cartilage cells growth. Presented work provides the
detailed examination of the impact of the
microencapsulation methods on the growth and viability
of chondrocytes. Potential application of the presented
work is the reconstruction of damaged joint cartilage.
Methods. The microcapsules, fibers or sheets were
obtained by three steps method. Alginate with cell was
gelled in calcium chloride solution, than it was coated
with chitosan and then again in the alginate solution of
ten times lower concentration then in the first phase.
Obtained forms were stored in the medium at 36
degrees. The influences of alginate concentration,
gelation time, type of chitosan, the form of microcapsule
and cell concentration on cells growth and viability were
investigated.
Results. The process of encapsulation does not destroy
the chondrocytes, the chosen method does not contain
harmful stages and creates conditions for their growth.
Application of encapsulated cells is slightly restricted by
the smaller growth rate as compared with that of free
cells. The growth and viability of chondrocytes are
significantly influenced by the size and the shape of
produced microcapsules but not by the type of chitosan
applied.
Conclusion. Considered method does not have any
limiting stage; all components which build the membrane
are
highly
biocompatible
and
capable
for
immunoisolation. Microcapsules coating make them
mechanically strong, the glucosamine compounds, which
are the products of enzymatic hydrolyzis exist naturally in
cartilage, and the alginate capability to reduced
dedifferentiation provides synthesis of appropriate type
of collagen. Thanks to all these advantages ACA system
appears to be a promising method for reconstruction of
damage cartilage.
Keywords. chondrocytes, cartilage, alginate, chitosan
(8.P23) HYALINE CARTILAGE REGENERATION BY
COMBINED THERAPY OF MICROFRACTURE AND LONGTERM BMP-2 DELIVERY
Yang HS (1), La WG (2), Bhang SH (2), Jang HK (3), Kim HJ
(4), Park JH (4), Kim BS (3)
1. Department of Bioengineering, Hanyang University,
Seoul; 2. School of Chemical and Biological Engineering,
Seoul National University, Seoul; 3. Interdisciplinary
Program of Bioengineering, Seoul National University,
Seoul; 4. Department of Orthopaedic Surgery, Ansan
Hospital, College of Medicine, Korea University, Ansan,
Republic of Korea
Introduction. Microfracture of cartilage induces
migration of bone marrow-derived mesenchymal stem
cells (BMMSCs) to cartilage defect sites. However, this
treatment often results in fibrocartilage regeneration.
Growth factors such as bone morphogenetic protein
(BMP)-2 induce the differentiation of BMMSCs into
chondrocytes, which can be used for hyaline cartilage
regeneration. Here, we tested the hypothesis that longterm delivery of BMP-2 to cartilage defects subjected to
microfracture would result in regeneration of high quality
hyaline-like cartilage, as opposed to short-term delivery
of BMP-2 or no BMP-2 delivery.
Methods. Rabbit articular cartilage defects were treated
with microfracture combined with one of the following:
no treatment, fibrin, short-term delivery of BMP-2,
Heparin-conjugated fibrin (HCF), or long-term delivery of
BMP-2. HCF and normal fibrin were used as carriers for
the long and short-term delivery of BMP-2, respectively.
Eight weeks after treatment, the cartilage regeneration
was evaluated by morphometrical analysis, histological
analysis, GAG contents analysis, and real-time polymerase
chain reaction(RT-PCR).
Results. Histological analysis revealed that the long-term
delivery of BMP-2 group (microfracture + HCF + BMP-2)
showed the most staining with alcian blue. A biochemical
assay, RT-PCR assay, and western blot analysis all
revealed that the long-term delivery of BMP-2 group had
the highest GAG content as well as the highest expression
level of collagen type II.
Conclusion. The long-term delivery of BMP-2 to cartilage
defects subjected to microfracture resulted in
regeneration of hyaline-like cartilage, as opposed to
short-term delivery or no BMP-2 delivery. This method
could be more convenient for hyaline cartilage
regeneration than autologous chondrocyte implantation
due to its less invasive nature and lack of cell
implantation. Since BMP-2 and microfracture are
currently in use clinically, this approach would be highly
feasible.
Acknowledgements. This study was supported by a grant
(No. 2010-0020352) from the National Research
Foundation of Korea
Keywords. Bone morphogenetic protein-2; Cartilage
regeneration; Heparin-conjugated fibrin; Microfracture
(8.P24) MESENCHYMAL STEM CELLS EXERT PARACRINE
EFFECTS ON OSTEOARTHRITIC CARTILAGE AND
SYNOVIUM.
Van Buul GM (1), Villafuertes E (1,2), Kops N (1), Bos PK
(1), Verhaar JAN (1), Weinans H (1,3), Bernsen MR (1), van
Osch GJVM (1)
1. Erasmus MC, Rotterdam, the Netherlands; 2. Hospital
Clínico San Carlos, Madrid, Spain; 3. Delft University of
Technology, Delft, the Netherlands
Introduction. Osteoarthritis (OA) is characterized by an
imbalance of anabolic and catabolic processes in synovial
joints, resulting in progressive cartilage damage.
Mesenchymal stem cells (MSCs) have recently been
discovered to have immunomodulatory capacities by
secreting several anti-inflammatory cytokines and growth
factors. MSCs are promising candidates for OA therapies,
although applied cells do not seem to actively participate
in formation of new cartilage. We studied the paracrine
effects of MSCs on OA cartilage and synovium explants in
vitro.
Methods. To stimulate primary human MSCs to secrete
immunomodulatory factors, they were cultured in
medium containing 10% FCS with additional TNFa and
IFNg (50 ng/ml each). After 24 hours medium was
collected and designated “conditioned medium”. Human
cartilage and synovium explants were cultured for 48
hours in conditioned medium or in unconditioned control
media with or without TNFa and IFNg (50ng/ml). Explants
were studied for expression of genes regulating
inflammation and extracellular matrix degradation.
Results. Cartilage: IFNg en TNFa upregulated ADAMTS-4
and IL-1RA in cartilage explants, while ADAMTS-5 and
MMP-13 were unaffected. Conditioned medium by MSCs
further upregulated IL-1RA and downregulated ADAMTS5 gene expression, whereas ADAMTS-4 and MMP-13
remained unchanged.
Synovium: IFNg and TNFa upregulated TNFa, IL-1b, IL-1RA
and SOCS1 in synovium. Conditioned medium
downregulated the cytokine-induced IL-1b expression and
further upregulated IL-1RA and SOCS1. MMP-13 gene
expression was not affected by IFNg and TNFa stimulation
or conditioned medium.
Conclusions. Conditioned medium containing factors
secreted by MSCs caused anti-catabolic and multiple antiinflammatory responses in our cartilage and synovium
explants. This indicates that MSCs have beneficial
paracrine effects on the metabolism of osteoarthritic
cartilage and synovium. These results offer a possible
working mechanism for MSCs to modulate the
osteoarthritis process, and encourage further use and
study of MSCs as a treatment for OA.
Keywords. Osteoarthritis, MSC, mesenchymal stem cell,
paracrine, cartilage
(8.P25) THE ANTI-OSTEOARTHRITIS DRUG, PENTOSAN
POLYSULFATE, STIMULATES BONE MARROW DERIVED
STRO
3+
MESENCHYMAL
PRECURSOR
CELL
PROLIFERATION, CHONDROGENIC DIFFERENTIATION
AND REDUCES APOPTOSIS WHEN CULTURED IN POROUS
COLLAGEN SCAFFOLDS
Ghosh P (1), Wu J (2), Shimmon S (2), Goldschlager T (3),
Zannettino A (4), Gronthos S (4), Jenkin G (5)
1. Mesoblast Ltd; 2. Institute of Nutraceutical research; 3.
Monash Medical Centre; 4. Hansen Institute; 5. Richie
Centre MIMR
Introduction. Our previous studies have shown that
Pentosan Polysulfate (PPS) stimulated Mesenchymal
Precursor Cells (MPC) chondrogenic differentiation in
micromass cultures. In this study we examine the ability
of PPS to induce MPC chondrogenesis, proliferation but
reduce apoptosis when seeded in commercial collagen
scaffolds.
Methods and Materials. MPC cells (70,000) were injected
into commercially available collagen sponges (Gelfoam or
OsseoFit) (2x6mm discs) then cultured in DMEM (+10%
FBS) supplemented with 0.0 - 20.0 ug/ml PPS for up to 21
days. In some cultures TGF-Beta-3 (0 - 20ng/mL) was
include in the absence or presence of PPS. Cell apoptosis,
viability and proliferation were monitored by
Tunnel/DAPTI /WST8 and 3H-Thymidine assays.
Proteoglycan (PG) synthesis was quantified by
incorporation of 35-S into sulphated glycosaminoglycans
normalised for cell number (DNA). MPC gene expression
was followed over days 7, 14 and 21 using real-time PCR
(RT-PCR).
Results. Bioassays showed that MPC viability and
proliferation was stimulated and apoptosis decreased by
PPS over 21 days. DNA synthesis was maximal with 2.5
ug/mL PPS (p < 0.03) on day 10. Proteoglycan
biosynthesis was maximal on day 10 (82% > control, p =
0.0005) with 2.5 ug/mL PPS, while 100% > control was
observed on day 14 at both 2.5 and 5.0 ug/mL PPS (p <
0.0001). TGF-Beta-3 induced maximal PG synthesis by
MPC at 10ng/mL (400%, p = 0.00001) but this effect was
enhanced synergistically to 650% in the presence of 5.0
ug/mL PPS (p = 0.00001). RT-PCR confirmed increased
expression of SOX-9, Aggrecan and type II collagen genes
at PPS concentrations of 2.5 - 10 ug/mL.
Conclusions. These studies confirmed that human MPC
cultured in collagen sponges in the presence of PPS
undergo proliferation and chondrogenic differentiation.
These data support the notion that PPS in combination
with MPC can be used for the repair of cartilage
osteochondral defects.
Keywords. Mesenchymal stem cells, chondrogenesis,
pentosan polysulfate, collagen scaffolds
(8.P26) PLATELET-DERIVED GROWTH FACTOR-AA IS A
POTENTIAL CHEMOATTRACTANT FOR MIGRATION OF
HUMAN BONE MARROW-DERIVED MESENCHYMAL
STEM CELLS IN ARTICULAR CARTILAGE INJURED
ATHYMIC RATS
Lee JM (1), Im GI (1)
1. Department of Orthopaedics, University of Dongguk
Ilsan Hospital
Cell motility is controlled by extracellular matrix
substrates and by secreted molecules such as chemokines
and growth factors. Cell migration in response to specific
external signals is termed chemotaxis. The understanding
of cell chemotactic mechanisms may lead to the novel
therapeutic strategies for tissue regeneration. The
purpose of this study is to investigate that molecules such
as chemokines and growth factors may direct BMSC
migration. For this, we used Boyden chamber assays to
select which chemokines or growth factors are able to
induce migration of human BMSC. BMSCs significantly
responded to several chemokines (IL-8, MIP-3a, SDF-1
and CCL2/MCP-1) and growth factors (PDGF-AA, HGF and
IGF-1). The most potent chemotactic effect was observed
with PDGF-AA compared to other chemoattractants. The
optimal response was observed at 50 ng/ml of PDGF-AA
in BMSC. The migration of human BMSCs reached peak
values at 593±123. Further the study was confirmed by
the expressions of chemoattractant receptors in
BMMSCs. It was found that the expression of CXCR2,
PDGFR-α, and c-Met were increased in serum-free
conditions, compared to the expressions of other
receptors. Interestingly, the expression level of PDGFR-α
was significantly increased than that of other
chemoattractant receptors. This study revealed that the
migration effect of BMSCs by PDGF-AA may be related to
the steady expression of PDGF receptor alpha in serumfree conditions and futher need to explore the receptor
signalling mechaism of PDGF in the chemotaxis of BMSC.
We also investigated that the selected migration factor
may effectively direct BMSCs in vivo nude rat model
through migration factor conjugated fibrin gel scaffolds.
In conclusion, this study demonstrates the ability of
human bone marrow-derived mesenchymal stem cells to
migrate in response to PDGF-AA, and probability of
cartilage repair in nude rat model. We suggest that PDGFAA conjugated fibrin gels are useful biomaterials for
injured-articular cartilage regeneration.
Keywords. human bone marrow-derived mesenchymal
stem cells, Migration, PDGF-AA, in vivo nude rat model
constructs. In this study we explore the feasibility of using
such a sandwich model.
Methods. A 4 mm layer of 3% agarose was allowed to gel
in a 12-wells plate. Afterwards, a ± 500 µm layer of 0.5%
agarose containing 50x106 bovine chondrocytes per ml
was added, and covered with another 2 mm layer of 3%
agarose (Fig 1). As a control 0.5% agarose discs containing
50x106 chondrocytes per ml were used. All constructs
were cultured in a) medium containing FBS (n=6 per
group) or b) serum-free medium with 10 ng/ml TGF-β3 (n
= 6 per group) for 32 days. All constructs were analyzed
for viability, biochemical content and matrix distribution.
Results. No significant differences in viability, matrix
content and distribution were observed between
standard agarose discs and the agarose sandwich model.
Discs and sandwiches cultured in presence of TGF-β3
contained significantly more proteoglycan and collagen
compared to those cultured in FBS-medium, and matrix
distribution was more homogeneous.
Conclusions. These results demonstrate that the agarose
sandwich model is suitable for use in cartilage tissue
engineering studies. The layered system did not limit
tissue development due to for instance an effect on
diffusion of matrix or nutrients. We will proceed to use
this model system for application of mechanical loading
to low-concentration agarose constructs.
Keywords. Agarose, cartilage, chondrocytes, TGF-B3
Schematic representation of the agarose sandwich model
9. CELL TRACTION: THE PROS AND
CONS IN VALVULAR AND VASCULAR
TISSUE ENGINEERING
(8.P27) OPTIMIZATION OF AN AGAROSE SANDWICH
MODEL FOR CARTILAGE TISSUE ENGINEERING
Kock LM (1), van Donkelaar CC (1), Gerrits CB (1), Ito K (1)
1. Eindhoven University of Technology
Introduction. The chondrocyte-seeded agarose model is a
well-established in vitro system used in cartilage tissue
engineering. Previously, we have shown that reducing
agarose concentration results in increased and more
uniform matrix production. Besides, it is known that
mechanical loading is an essential trigger for stimulation
of cartilage growth. However, direct mechanical loading
of low-concentration agarose constructs is impossible,
because initially these are weak and brittle. Seeding cells
in a low-concentration agarose layer in between stiffer
agarose layers (‘agarose sandwich’) would enable to
apply mechanical loading to low-concentration agarose
Chair: Anita Driessen-Mol
Co-chair: Stefan Jockenhoevel
Keynote speaker: Stefan Jockenhoevel
Organizers: Anita Driessen-Mol, Stefan Jockenhoevel
Synopsis: Valvular and vascular tissue engineering rely on
extracellular matrix production by cells seeded into a
degrading scaffold material. The seeded cells adapt a
myofibroblast phenotype, characterized by synthetic as
well as contractile activity, and naturally exert traction
forces to their surroundings. In nature, these
surroundings are capable of withstanding these forces by
the hemodynamic environment the tissue is in (e.g.
pressure), the degree of constraint of the tissue (e.g.
blood vessels are constrained in axial direction), and the
extracellular matrix properties (both composition and
mechanical behaviour).
Tissue engineering has made us realize how delicate this
balance in nature is. Cell traction on the one hand is
shown beneficial for tissue maturation and alignment in
engineered tissues, while on the other hand is causing
loss of shape. Unbalance in the extracellular matrix
properties, hemodynamics and cell traction in engineered
heart valves was demonstrated to result in leaflet
shrinkage, a problem commonly observed in animal
studies, and resulted in regurgitation of the valve and loss
of function. This symposium offers a platform to discuss
the pros and cons of cell traction in valvular and vascular
tissue engineering. We hope to share insights on its
fundamentals and to work towards fine-tuning of this
delicate balance between cell traction, extracellular
matrix properties and hemodynamics towards functional
valvular and vascular tissue engineering.
(9.KP) CELL TRACTION: THE PROS AND CONS IN
VALVULAR AND VASCULAR TISSUE ENGINEERING
Jockenhoevel S (1), Driessen-Mol A (2)
1. Department of Tissue Engineering & Textile Implants,
AME-Helmholtz Institute of the RWTH Aachen University,
Aachen, Germany; 2. Department of Biomedical
Engineering, Eindhoven University of Technology,
Eindhoven, The Netherlands
Valvular and vascular tissue engineering rely on
extracellular matrix production by cells seeded into a
degrading scaffold material. The seeded cells adapt a
myofibroblast phenotype, characterized by synthetic as
well as contractile activity, and naturally exert traction
forces to their surroundings. In nature, these
surroundings are capable of withstanding these forces by
the hemodynamic environment the tissue is in (e.g.
pressure), the degree of constraint of the tissue (e.g.
blood vessels are constrained in axial direction), and the
extracellular matrix properties (both composition and
mechanical behaviour). Tissue engineering has made us
realize how delicate this balance in nature is. Cell traction
on the one hand is shown beneficial for tissue maturation
and alignment in engineered tissues, while on the other
hand is causing loss of shape.
The keynote lecture will give an overview on the
physiological and pathophysiological mechanisms of
tissue shrinkage in general and specifically the relevance
of these factors on cardiovascular tissue engineering.
Furthermore the three major factors of tissue engineering
(1) cells, (2) scaffolds and (3) stimuli will be analyzed with
regard to their influence on tissue retraction.
(9.O1) ENDOGENOUS TISSUE CONTRACTILITY SPATIALLY
REGULATES THE VEGF SIGNALING AND ANGIOGENESIS
IN SELF-ORGANIZING MICROFABRICATED TISSUES
Rivron N (1), Vrij E (2), Rouwkema J (2), Truckenmuller R
(2), Le Gac S (2), Van den Berg A (2), Van Blitterswik C (2)
1. University of Twente, Hubrecht Institute; 2. University
of Twente
Endogenous physical forces can drive the organization of
tissues (1-2). The underlying mechanisms are currently
based
on
cell
surface
mechanics
(3)
or
mechanotransduction (4) and are thus separated from
known conserved mechanisms including the formation of
morphogen gradients. Here using an array of
autonomously contracting and deforming, 3D,
microfabricated, tissues, we show that tissue geometry
and endogenous contractility spatially regulates the
Vascular Endothelial Growth Factor (VEGF) signaling and
the local formation of vascular patterns. The
microfabricated
tissues
stereotypically
and
heterogeneously changed shape, compacted and formed
robust patterns of vascular structures in regions of high
deformation. This emergence correlated with the local
over-expression of the receptor VEGFR2 and with the
formation of a tissue-scale gradient of VEGF. We propose
that endogenous tissue contractility and deformation is a
morphogenetic regulator of angiogenesis, a finding which
should stimulate new therapeutic strategies for vascular
diseases and regenerative medicine.
References.
1. Mammoto T & Ingber DE (2010) Development
137(9):1407-1420.
2. Wozniak MA & Chen CS (2009) Nat Rev Mol Cell Biol
10(1):34-43.
3. Lecuit T & Lenne PF (2007) Nat Rev Mol Cell Biol
8(8):633-644.
4. Mammoto A, et al. (2009) Nature 457(7233):11031108.
Keywords. vascular pattern, endogenous contractility,
VEGF signaling
(9.O2) THE POTENTIAL OF PROLONGED TISSUE CULTURE
TO REDUCE STRESS GENERATION AND RETRACTION IN
ENGINEERED HEART VALVE TISSUES.
Van Vlimmeren MAA (1), Driessen-Mol A (1), Oomens
CWJ (1), Baaijens FPT (1)
1. Eindhoven University of Technology
Introduction. Tissue engineered heart valves develop a
good tissue architecture, induced by traction forces,
when cultured constrained. However, during culture cell
traction causes tissue compaction, resulting in leaflet
flattening. At time of implantation, the leaflets have to be
separated and cell traction causes leaflet retraction. To
get insight into these mechanisms and to develop
solutions, we have developed an in vitro model system to
quantify and correlate stress generation, compaction,
retraction and tissue quality during a prolonged culture
period of 8 weeks.
Methods. PGA/P4HB strips were seeded with vascularderived cells and cultured for 4, 6 and 8 weeks (n=5 per
time point). Compaction in width was measured during
culture, while stress generation and retraction in length
were measured after culture when constraints were
released. Further, the amount of DNA, GAG, collagen and
collagen cross-links was assessed.
Results. Compaction started after 2 weeks and continued
up to 66.2±1.7% at week 4, after which width remained
constant (fig 1A). Stress generation reduced from
11.8±0.9 kPa at week 4 to 2.4±0.4 kPa at respectively
week 8 (fig 1B). Tissue retraction reduced from 44.0±3.7%
at week 4 to 26.1±2.2% at week 8 (fig 1C). The reduced
stress generation over time correlated with the reduced
retraction. The amount of DNA, collagen and collagen
cross-links was constant at all time points. The amount of
GAGs was increased at week 6 and 8 compared to week 4
and correlated to the reduced stress generation.
Conclusion. In summary, increasing culture time resulted
in decreased stress generation and retraction, likely as a
result of the increased amount of GAGs. These results
demonstrate the potential of prolonged tissue culture in
developing functional, non-retracting, TE heart valves.
Acknowledgement. The authors gratefully acknowledge
the support of the Smart Mix Program of the Netherlands
Ministry of Economic Affairs and Education, Culture and
Science.
Keywords. compaction, retraction, stress generation,
heart valve tissue
(9.O3) EFFECT OF CROSSLINKING OF FREEZE-DRIED AND
CRITICAL POINT DRIED COLLAGEN SCAFFOLDS ON
PHYSICAL PROPERTIES AND CELL FUNCTION: RELEVANCE
FOR HEART VALVE TISSUE ENGINEERING
Carubelli I (1), Tseng YT (2), Sarathchandra P (1),
Czernuszka JT (2), Chester AH (1), Yacoub MH (1), Taylor
PM (1)
1. Imperial College London, National Heart and Lung
Institute, Heart Science Centre, Harefield Middx, UB9 6JH
UK; 2. Department of Materials, University of Oxford,
Parks Road, Oxford OX1 3PH, UK
Introduction. Suitable cell-scaffold constructs for tissue
engineering a heart valve should be mechanically strong
and compatible with cells, allowing them to grow, secrete
appropriate extracellular matrix components (such as
collagen,
elastic
fibres
and
proteoglycans/glycosaminoglycans) and not promote
calcification. Collagen based scaffolds have several
desirable characteristics. However, optimal processing
methods have not been established.
Methods. We have evaluated the cellular compatibility
and physical properties (thermal stability, resistance to
enzymatic degradation, Young’s modulus, pore size and
permeability) of 4 different collagen scaffolds. Scaffolds
were manufactured using freeze drying (FD) or critical
point drying methods (CPD) and either physically
crosslinked with dehydrothermal treatment (DHT) or
chemically
crosslinked
with
1-ethyl-3(3-dimethyl
aminopropyl)
carbodiimide/N-hydroxysuccinimide
(EDC/NHS). Cell compatibility was studied using a
dynamic seeding process with human mesenchymal stem
cells (MSCs) and following cell proliferation and cell
phenotype.
Results. Chemical crosslinking proved to increase scaffold
resistance and decrease its permeability better than
physical crosslinking. All scaffolds were compatible with
MSCs as judged by proliferation of the cells and their
ability to produce extracellular matrix and not to
differentiate towards osteogenic, chondrogenic or
endothelial lineages. FD scaffolds with EDC/NHS
crosslinking were the only scaffolds able to withstand
pressures up to 80 mmHg and showed the highest
Young’s modulus compared to the other scaffolds.
Conclusions. Our results suggest that FD EDC/NHS
scaffolds are good candidates for heart valve tissue
Keywords. Collagen scaffold, heart valve tissue
engineering, Extracellular matrix, Mesenchymal stem cell
(9.O4) EFFECT OF MECHANICAL CONDITIONING ON CELLMEDIATED TISSUE CONTRACTION IN FIBRIN-BASED
TISSUE ENGINEERED HEART VALVES
Hasken S (1), Kreitz S (1), Schmitz-Rode T (1), Mela P (1),
Jockenhoevel S (1)
1. Dept. of Tissue Engineering & Biomaterials, Institute of
Applied Medical Engineering Helmholtz Institute of the
RWTH Aachen University, Germany
Introduction. Functional valvular tissue engineering aims
at developing living tissue by seeding or embedding cells
into degradable scaffolds which are mechanically
conditioned in bioreactors to reach functionality prior to
implantation. The presence of cells is responsible for
production of extracellular matrix and reorganization of
fibers. However, it also causes contraction of the tissue
and consequent changes of its 3D geometry, which in the
case of heart valves results in insufficiency as commonly
observed in vivo and in vitro. We applied different
conditioning protocols to fibrin-based 3 leaflet-heart
valves in bioreactors and evaluated their effects on leaflet
retraction by ultrasound.
Methods. The fibrin gel valves were produced using a
previously reported moulding technique by mixing a
fibrinogen solution (20mg/ml) with TBS containing ovine
carotid artery-derived cells, CaCl2 and thrombin. The cell
concentration was 10X106/ml of the final volume.
After moulding two different conditioning strategies were
followed in custom made bioreactors: 1) dynamic
conditioning and 2) static conditioning on the mould
followed by dynamic conditioning. Ultrasound images
were taken to evaluate leaflet configuration and function.
Results. The heart valves conditioned only dynamically
were able to open and close in a satisfactory way at the
very beginning of the protocol but showed clear
shrinkage of the leaflets in the following days till no
leaflet could be detected anymore (Fig.1a). The valves
receiving static and dynamic conditioning showed a much
reduced leaflet retraction and good functionality (Fig.1b).
Conclusions. Optimization of the conditioning protocol is
one crucial step towards the development of functional
tissue engineered heart valves. The capability to drive and
actively modulate the production of the extra cellular
matrix is particularly important to reach adequate
functionality.
Acknowledgements.
The
authors
thank
the
Fördergemeinschaft Deutsche Kinderherzzentren e.V. for
financial support.
Keywords. fibrin, heart valve, mechanical stimulation,
tissue contraction
effect of oxidized LDL will be investigated to further test
this hypothesis.
Acknowledgments. This work is funded by the Irish
Research Council for Science Engineering and Technology
Keywords. Atherosclerosis, Stem cells, Chondrogenesis,
Endochondral ossification
(9.O5) VESSEL DERIVED STEM CELLS CONTRIBUTE TO
ENDOCHONDRAL OSSIFICATION OF ATHEROSCLEROTIC
PLAQUE
Leszczynska A (1), O’Doherty A (2), Barry F (1), O’Brien T
(1), Murphy M (1)
1. Regenerative Medicine Institute, National University of
Ireland, Galway, Ireland; 2. National Centre for Biomedical
Engineering Science, National University of Ireland,
Galway, Ireland
Introduction. Pericytes, although traditionally considered
as supporting cells, have recently been proposed to have
a more active role in the repair and pathogenesis of
various vascular diseases. There is growing body of
research work indicating that the vessel wall contains a
number of progenitor cell niches that remain as yet
completely defined. In this study, we hypothesized that a
pericyte-like stem cell population, termed vessel derived
stem cells or VSCs with chondrogenic and osteogenic
potential exists in the vessel wall and in presence of the
inflammatory cytokines seen in atherosclerotic
environment, contributes, along with the circulating
mesenchymal stem cells to the calcification of
atherosclerotic plaque which occurs through the
endochondral pathway.
Methods. VSCs from aortae of ApoE-/- mice and
background C57BL/6 mice were isolated and
characterized for cell surface markers by flow cytometry
and immunocytochemistry. MSCs from bone marrow of
these mice were also isolated and characterized.
Chondrogenic potential of these cells was investigated in
presence or absence of inflammatory cytokines such as IL6 and IFN-γ. Real time PCR was performed to analyze the
up- or down-regulation of key factors in chondrogenic
pathway.
Results and Discussion. VSCs were strongly positive for
Sca-1, CD44 and negative for CD31 and CD34.
Immunocytochemistry for specific pericyte marker 3G5
revealed that a sub-population of VSCs expressed 3G5
(Figure 1). Differentiation assays demonstrated the ability
of the cells to differentiate into bone and cartilage. VSCs
had significantly higher GAG/DNA ratio than MSCs
indicating increased chondrogenesis. That both MSCs and
VSCs from the ApoE-/- atherosclerotic mice generate a
more mature hypertrophic chondrocyte than cells from
the C57BL/6 mice is interesting and suggests that the
atherosclerotic environment may modulate the stem cell
phenotype. Col-type II and aggrecan expression and
Figure 1. 3G5 staining.
(9.O6) CLAY-GELS CAN LOCALIZE VEGF AND INDUCE
ANGIOGENESIS IN VITRO AND IN VIVO
Dawson JI (1), Kanczler JM (1), Yang XB (2), Attard GA (3),
Oreffo ROC (1)
1. University of Southampton, School of Medicine; 2.
University of Leeds, School of Dentistry; 3. University of
Southampton, School of Chemistry
Introduction. Hydrogels offer considerable potential as
tissue engineering matrices, however their essential
hydrophilicity presents challenges for the retention, in
space and time, of bioactive molecules. Certain clays are
known for their ability to adsorb biological molecules due
to the large and highly charged specific surface area of
the nano/micro-sized particles. We show the potential of
a synthetic smectite clay suspension to self-organise,
encapsulate viable cells and localise exogenously applied
angiogenic factors inducing an angiogenic response in
vitro and in vivo.
Methods. Suspensions of laponite, a synthetic smectite,
were added drop-wise to cell culture media containing
model protein (albumin, lysozyme) or vegf165. Protein
diffusion and uptake by laponite capsules was assessed by
assaying supernatant using bradford assays and elisas, or
confocal analysis via flouroprobe labeling. In vitro
angiogenic induction by laponite-bound vegf was
assessed using the human umbilical vein endothelial cell
(huvec) tubule-formation assay. For in vivo
characterization, laponite encapsulated collagen scaffolds
were incubated in vegf media for 2hrs before
implantation in a murine femoral defect model. Neoangiogenesis was quantified via micro-ct.
Results. Upon addition to physiological saline, freeflowing laponite suspensions self-organized into stiff gels
allowing encapsulation of cells, matrix proteins and
growth factors. While negligible diffusion of protein out
of laponite capsules was observed over 14 days, rapid and
extensive uptake and binding of protein by laponite
capsules was observed. To test the bioactivity of laponitebound protein, the effect of laponite-bound vegf on
huvec tubule-formation was assayed. Laponite films
exposed to vegf for two hours before washing yielded
equivalent tubule-organization to positive controls. In
vivo
studies
revealed
significantly
enhanced
vascularisation compared to controls (fig 1).
Conclusion. This work describes a novel clay-gel based
strategy for the delivery and application of growth factors
without the need for complex chemical modifications
thus offering significant potential for the delivery of
regenerative microenvironments.
Keywords.
hydrogels,
growth
factor
delivery,
angiogenesis, clays
(9.O7) ADVANTAGES OF DENUDED HUMAN UMBILICAL
VEIN (HUV) OVER DECELLULARIZED HUV AS SCAFFOLD
FOR VASCULAR TISSUE ENGINEERING
Mangold S (1), Schrammel S (2), Bursa J (3), Huber G (4),
Bronger H (5), Schmid C (1), Hoenicka M (1)
1. University of Regensburg Medical Center, Department
of Cardiothoracic Surgery, Regensburg, Germany; 2.
University of Applied Sciences Regensburg, FB
Maschinenbau, Regensburg, Germany; 3. Brno University
of Technology, Institute of Solid Mechanics, Mechatronics
and Biomechanics, Brno, Czech Republic; 4. University of
Regensburg, Krankenhaus Barmherzige Brüder, Klinik St.
Hedwig, Regensburg, Germany; 5. OB/GYN, Klinikum
rechts der Isar, Munich, Germany
Objective. To compare the utility of endotheliumdenuded and completely decellularized human umbilical
veins (HUV) as scaffolds for tissue-engineered smallcaliber vessel grafts.
Methods. HUV were endothelium-denuded by luminal
dehydration (60 ml/min carbogen) or decellularized using
(1) a detergent mixture (Triton X-100, sodium
deoxycholate, IGEPAL-CA630, 0.025% each), (2)
peroxyacetic acid (0.1 %), or (3) alternating washes with
3M NaCl and distilled water, each followed by nuclease
treatment and extensive washing. Scaffold compositions
were analyzed by histology and immunohistology. Failure
stresses were determined in a tensile testing rig. Calcein
AM stained HUVEC were seeded on the scaffolds at
densities of 5E5 cells/cm2.
Results. Denudation removed endothelial cells without
damaging other wall components or decreasing
tetrazolium dye reduction. Decellularization caused an
almost complete loss of H&E-stainable material.
Remnants of degenerate nuclei were removed by
nuclease treatment. In contrast to denudation,
decellularization caused a loss of laminin and fibronectin
staining, as well as fragmentation of elastic fibers. Failure
stresses were not decreased by denudation or by
chemical treatments, but by nuclease treatment and
were extrapolated to burst pressures of 2160 mm Hg
(native), 1880 mm Hg (denuded), and 1580 mm Hg
(decellularized). Static HUVEC seeding resulted in a
confluent neoendothelium on denuded vessels after 3
days culture. Decellularized vessels showed incomplete
coverage on day 1 and a loss of viable cells until day 3.
Seeding of denuded HUV in a perfusion bioreactor
resulted in a flow-resistant neoendothelium.
Discussion. Denuded HUV maintain a metabolically active
smooth muscle layer and provide a superior surface for
endothelial cell seeding compared to decellularized HUV.
This may be attributed to the preservation of intact
basement membranes. Therefore denuded HUV are to be
preferred to decellularized HUV for vascular tissue
engineering.
Acknowledgements. This study was funded by Deutsche
Forschungsgemeinschaft (BI 139/2-1, HA 4380/5-1, and LI
256/68-1).
Keywords. endothelium; decellularization; scaffold;
umbilical vein
(9.P1) INFLUENCE OF ADDITIONAL NORMOBARIC
HYPOXIA ON EXERCISE INDUCED HEMATOPOIETIC STEM
CELL RELEASE
Kröpfl JM (1), Pekovits K (2), Stelzer I (3), Sedlmayr P (2),
Groeschl W (1), Hofmann P (1,4), Domej W (1,5), Dohr G
(2), Müller W (1)
1. HPR Graz (KFU & Med Uni Graz); 2. Institute of Cell
Biology, Histology and Embryology (Med Uni Graz); 3.
Clinical Institute of Medical and Chemical Laboratory
Diagnostics; 4. Institute of Sport Sciences (KFU Graz); 5.
Institute of Pulmonology (Med Uni Graz)
Introduction. The release of adult hematopoietic stem
cells (HSCs) was shown to improve regeneration
processes in the human body. Recent data suggest that
an elevated level of HSCs in the peripheral blood supports
tissue renewal and patients recovery. Both physical
exercise and normobaric hypoxia may act as triggers for
HSC mobilization. The aim of our study was to investigate
the effect of normobaric hypoxia and physical exercise on
the release of HSCs from the bone marrow into the
circulation.
Methods. Six healthy male subjects (26,5 +/- 5,1 yrs)
underwent a standardized incremental exercise test (40
W+20 W/min) under either normoxic (FiO2 ~ 0.21) or
hypoxic conditions (FiO2 < 0.15, equals 3.500 m, 3 h
exposure) within a time span of at least one week. Blood
was drawn from the cubal vein before and 10, 30, 60 and
120 min after the exercise. The number of HSCs in the
peripheral blood was analyzed by means of flow
cytometry (CD 34/ CD 45 positive cells). Standard markers
of exercise performance (Pmax, VO2max, Lamax, HRmax)
were obtained.
Results. The physical challenge of the incremental test
showed a significant increase of HSC release under
normoxic as well as hypoxic conditions (repeated
measures ANOVA using Fisher’s LSD, p < 0.05; Fig.1) after
10 min of recovery. There was not any significant
difference detectable between normoxia and hypoxia
regarding the HSC level (p > 0.05).
Conclusions. The results of this study indicate that the
HSC release to the peripheral blood is induced by
intensive exercise under both hypoxic and normoxic
conditions, but there was no greater effect on circulating
HSC numbers by addional hypoxia. We may suggest that
the short term hypoxic exposure of 3 h at approx. 3.500
m simulated altitude does not have any (additional) effect
on HSC mobilization from the bone marrow.
Keywords. hematopoietic stem cells, exercise, hypoxia,
facs analysis
(9.P2) HYBRID POLYMERIC IPN SCAFFOLDS FOR CARDIAC
HEART VALVE TISSUE ENGINEERING
Martínez-Crespiera S (1), Fernández N (1), Herrero M (1),
González S (1), Rodríguez C (1), Saint-Pierre G (1)
1. PERA
Introduction. Despite the efforts made in order to
improve the mechanical and biological performance of
the current clinical available mechanical and
bioprosthetic heart valves, the ideal prosthesis has not
been yet developed. Tissue engineering appears as a
promising alternative to overcome the main drawbacks of
the existent prosthesis (non-obstructive, nonthrombogenic, biocompatible and long-term lasting).
However, due to the technical difficulties of an efficient
heart valve prosthesis design, to date no tissue
engineered heart valve has demonstrated to be
successful at clinical level. Bioscent project (see
acknowledgment) aims at developing interpenetrating
network (IPN) of natural and synthetic polymers to
provide a novel generation of scaffold for heart valve
tissue engineering.
Materials and Methods. IPN of natural (sodium alginate,
chitosan) and synthetic polymers (PVA) are prepared.
Solvent casting is used to produce the polymeric blends.
Results. Solvent casting of the IPN systems offers
homogeneous, flexible and biocompatible scaffolds. The
mechanical properties have been validated with a
developed aortic valve FEM (Finite Element Method)
model. Through this model it has been demonstrated
how these IPN scaffolds are suitable materials that enable
the development of a tissue engineering autologous heart
valve compliant with the behaviour of the native valve.
Moreover it has been proved that mechanical properties
are not affected by the presence of calcium ions.
Conclusions. In the present work hybrid IPN for heart
valve scaffolds are presented. These systems are
biocompatible and present suitable mechanical
properties for the tissue engineering of the heart valves.
Acknowledgments. The present work is carried out in the
scope of BIOSCENT. Project full title: “BIOactive highly
porous and injectable Scaffolds controlling stem cell
recruitment, proliferation and differentiation and
enabling angiogenesis for Cardiovascular Engineered
Tissues” funded by European Commission FP7 Program
under Grant agreement no.: ID214539.
Disclosures. The present results are property of the
Bioscent Consortium.
Keywords. interpenetrating network (IPN), scaffold,
polyvinyl alcohol (PVA), sodium alginate (SA), solvent
casting
(9.P3) DEVELOPMENT OF NOVEL TISSUE ENGINEERED
SMALL DIAMETER VASCULAR GRAFT USING TRIMER
PEPTIDE
Narita Y, (1), Kuwabara F (1), Yamawaki-Ogata A (1), Kanie
K (2), Kato R (2), Satake M (3), Kaneko H (3), Honda H (2),
Ueda Y (1)
1. Department of Cardiac Surgery, Nagoya University
Graduate School of Medicine; 2 Department of
Biotechnology, Nagoya University Graduate School of
Engineering; 3. Technology Innovation Center, Teijin
Limited
Introduction. Both rapid endothelialization and the
prevention of intimal hyperplasia are essential to improve
the patency of small-diameter vascular grafts (SDVGs).
Using the peptide array-based screening system, we
identified the peptide “Cysteine-Alanine-Glycine (CAG),”
which has a high affinity for endothelial cells and a low
adhesive property for smooth muscle cells. It is known
that thrombosis contribute early stage occlusion, and
intimal hyperplasia contributes to late stage occlusion of
the SDVG. Meanwhile, thrombosis is caused by luck of
endothelium, and intimal hyperplasia is caused by
excessive synthesis of extracellular matrix from
dedifferentiated smooth muscle cells. In this study, we
report an in vivo analysis of the novel SDVGs that were
constructed with a biodegradable polymer (poly-εcaprolactone) containing CAG peptide in rat.
Methods. The novel tissue engineered (TE)-SDVG, which
measured 0.7 mm in diameter and 7 mm in length, was
fabricated using the electrospinning technique. The
carotid arterial replacement was performed on SpragueDawley rats using the SDVGs with (group CAG) or without
CAG (group C). Histological and biochemical assessments
were performed at 1, 2 and 6 weeks after implantation.
Results. The ratio of endothelialization was significantly
higher in group CAG compared to group C (CAG vs C:
64.4±20.0% vs 42.1±8.9% at 1 week p=0.02, 98.2±2.3% vs
72.7±12.9% at 2 weeks p=0.001, and 97.4±4.6% vs
76.7±5.4% at 6 weeks, p<0.001). Additionally, Western
blot analysis showed that the intensity of the endothelial
nitric oxide synthase at 1 week of group CAG was
significantly higher than that of group C (CAG vs C:
1.20±0.37 vs 0.34±0.16, p=0.01), and that α-smooth
muscle actin at 6 weeks in group CAG was significantly
lower than that of group C (CAG vs C: 0.89±0.06 vs
1.25±0.22, p=0.04).
Conclusions. Our developed TE-SDVG with CAG promoted
rapid endothelialization and potential to inhibition of
intimal hyperplasia.
Keywords. small diameter vascular graft, electrospinning,
peptide
10. CELL VIABILITY AND TISSUE
BANKING
Chair: Blanca Miranda
Co-chair: Antonio Fernández-Montoya
Keynote speaker: Alice Warley
Organizers: Blanca Miranda, Salvador Oyonarte
Synopsis: Construction of artificial tissues and organs by
tissue engineering is one of the fields of medical research
that has experienced major progress in recent years. In
this regard, and to ensure the appropriate function of the
developed organs, an accurate evaluation and quality
control of the constructed tissues and organs is very
important, especially if these are generated and stored in
Tissue Banks for clinical uses.
Evaluation of the suitability of the developed tissues and
organs has to be carried out at different levels and using
different techniques. In the first place, the researcher
must evaluate the viability of the primary cell cultures
that will be used to generate the tissue constructs, since
only viable cells are suitable for clinical use. In this regard,
the development of extremely sensitive techniques like
the electron probe X-ray microanalysis allows the
scientist to not only evaluate the viability of the cells in
the culture, but also to predict the short and long-term
behaviour of these cells. On the other hand, evaluation of
the constructed tissue substitutes have to ensure that
both the structure and the function of the constructs is
adequate and that these tissues and organs are similar to
the normal, native tissues that the researcher pretends to
reproduce in vitro. In this milieu, long-term storage of
bioengineered tissues in tissue banks is highly dependent
on the use of several cryoprotection agents. However,
most preservation protocols are associated to certain
degree of loss of cell viability or structural tissue damage.
For that reason, evaluation of cell viability and tissue
structure is especially important for tissues stored in
tissue banks.
All kind of works focused on the evaluation of cell viability
of cells and tissues generated by tissue engineering and
on tissue banking are welcome to this symposium,
including methods and techniques based, among others,
on:
- Dye exclusion tests.
- Intracellular components release.
- Metabolic and functional tests.
- Electron-probe X-ray microanalysis.
- Colony formation assays.
- In vitro and in vivo analyses.
- Gene expression analyses.
- Cryoprotection.
- Tissue storage.
- Vitrification.
- Tissue banking.
(10.KP) X-RAY MICROANALYSIS IN THE STUDY OF CELL
VIABILITY
Warley A (1)
1. CUI, King’s College London
For cell culture studies and for tissue engineering the
ability to assess cell viability is important. Whereas
traditional dye exclusion methods are routinely used in
tissue culture, they are difficult to adapt for tissue
engineering and also have the added disadvantage that
they monitor rather than predict cell death. The ratio of
K/Na has proved to be a reliable indicator both of cell
viability and of cell vitality, and distinguishes between
apoptotic and necrotic cell death pathways. Here I will
review the use of X-ray microanalysis, an electron
microscopy technique that allows the detection of
element content in cells and tissues provided that
suitable preparation procedures have been followed, for
the study of cell viability/vitality in cell cultures, and
suggest how this technique might be adapted to study
viability in tissue constructs.
(10.O1)
MATERIALS
CHARACTERISATION
AND
MESENCHYMAL STEM CELL RESPONSE ON PLCL
MATERIALS
Barron V (1), Rooney N(2), Barry F (1), Murphy M (1)
1. NUI, Galway; 2. Proxy Biomedical Ltd
To date, a range of biomaterials have been employed to
create scaffolds for a great variety of tissue engineering
applications. Previous research has shown that the
biological response of cells on tissue-engineered scaffolds
depends on the surface topography, chemical
composition and mechanical properties of the construct.
As a consequence, it is important to develop a deep
understanding of the materials properties and cell
response. To this end. two poly(l-lactide--caprolactone)
(PLCL) materials with the same chemical composition but
different surface topographies were investigated. As
expected, there was no difference in the chemical
composition of the two PLCL materials with characteristic
peaks at 1750cm-1 for C=O, 1180–1080cm-1 for C-O-C
and 1041cm-1 for CH3 functional groups. With respect to
polymer morphology, both materials exhibited a glass
transition temperature (Tg) of 17°C, with no crystalline
melt peaks, indicative of an amorphous blend. As seen
previously [1], the mechanical properties were altered as
a result of the surface features (Figure 1), with values of
1.39MPa and 2.02MPa recorded for the pitted and
porous materials, respectively. To investigate cell
response, human mesenchymal stem cells (MSC) were
seeded at a density of 20,000 cells/cm2 and maintained
for 24 hours at 37°C in a humidified atmosphere of 5%
CO2 at 37˚C. PLCL strips, 1/10 of the total area of the well,
were placed directly on the cells and incubated for an
additional 24 hours. Using a Guava Cytosoft cell sorter, it
was determined that there was statistical difference in
the cell viability of cells grown in the presence of the PLCL
materials when compared to the controls on TCP. In
summary, the data presented herein gives a deeper
insight into the materials properties and the cell response
of two PLCL materials and as such is the first step in
characterizing these PLCL substrates as delivery vehicles
for MSC.
References. 1. McGlohorn J.B. et al. Tissue Eng..10:505
2004.
Acknowledgments. The authors would like to thank the
Science Foundation Ireland (09/SRC/B1794) for providing
financial support to this project.
Keywords. PLCL scaffolds, materials characterisation,
stem cell response
(10.O2) MEASURING CELL VIABILITY IN 3D SCAFFOLDS
USING CONFOCAL MICROSCOPY
Dittmar R (1), Potier E (1), Van Zandvoort MAMJ (2), Ito K
(1)
1. Department of Biomedical Engineering, Eindhoven
University of Technology, Eindhoven, The Netherlands; 2.
Department of Biomedical Engineering, Cardiovascular
Research Institute Maastricht, Maastricht University,
Maastricht, The Netherlands.
Introduction. Cell viability (CV) is an important parameter
to evaluate the effect of environmental conditions on cell
behavior, yet current assays are rather invasive. Recently,
we demonstrated that two-photon microscopy could
accurately assess CV in situ in three-dimensional (3D)
scaffolds without staining based on differences in autofluorescence emission spectra of live and dead cells.
However, two-photon microscopy requires more
specialized equipment. Therefore, the objective of this
study was to evaluate confocal microscopy as a noninvasive tool to assess CV, in a similar fashion in 3D
collagen gels.
Methods. Mixtures of live and dead C2C12 myoblasts
(0%, 25%, 50%, 75%, and 100% live cells) were prepared
and CV was determined using the trypan blue (TB) assay.
Cell seeded collagen gels (CSCG, n=5/cell mixture) were
produced by mixing collagen solution with the live/dead
cell mixtures (3.5x106 cells/CSCG). After polymerization,
two
consecutive
confocal
microscopy
images
(λexc=458nm) of the CSCG were acquired through
bandpass filters of 475-525nm and 560-615nm,
respectively (n=30 images/CSCG). An intensity ratio per
imaged cell was calculated as averaged intensity from
image 1/averaged intensity from image 2. Receiver
operating characteristic (ROC) analysis was performed to
calculate a threshold ratio for cell differentiation.
Results. Ratios of 100% live and dead cells were
significantly different and a threshold ratio of 0.68 was
determined (Fig.1A). Applying this threshold, no
significant differences between the TB assay and confocal
microscopy were found in measuring CV (Fig.1B).
Nevertheless, CV values acquired with confocal
microscopy showed no significant differences between
0% and 25%, and 75% and 100% targeted CVs, whereas all
TB viability groups were significantly different.
Conclusion. The results demonstrate that autofluorescence intensity as measured by confocal
microscopy can be used to assess CV in 3D scaffolds.
However, it appears to be less sensitive to constructs with
mostly alive or dead cells.
Funding. EU-FP7 consortium Genodisc
References. [1] Dittmar. Trans Orthop Res Soc 2010, (35)
(10.O3)
EFFECTS
OF
CRYOPRESERVATION
ON
PERIPHERAL BLOOD MONONUCLEAR CELLS AND
ENDOTHELIAL PROGENITOR CELLS
Sofrenovic T (1), McEwan K (2), Suuronen EJ (1), Kuraitis D
(1).
1. University of Ottawa Department of Cellular and
Molecular Medicine; University of Ottawa Heart Institute;
2. University of Ottawa Heart Institute.
Introduction. Regenerative medicine has become an
appealing therapeutic method; however, stem and
progenitor cells are not always freshly available.
Cryopreservation offers a way to freeze the cells as they
are generated, for storage and transport until required
for therapy. Nevertheless, the effects of cryopreservation
on the cells, in this case endothelial progenitor cells
(EPCs) shown to be involved in neovascularization, have
not been extensively studied.
Methods. Peripheral blood mononuclear cells (PBMCs)
were extracted from healthy donors (n=6) using density
gradient centrifugation. The freshly isolated cells were
either analyzed or frozen with liquid nitrogen in media
containing 6% plasma serum and 5% dimethyl sulfoxide.
After being frozen for 1 day (early) or 28 days (late), the
PBMCs were thawed and analyzed or cultured on
fibronectin with endothelial basal media for 4 days to
generate EPCs. Analysis of the cells consisted of flow
cytometry, for viability and various progenitor and stem
cell surface markers, as well as functional assays for the
adhesion and migration potential.
Results. The viability of PBMCs decreased after
cryopreservation (p<0.01). CD34 and VEGFR2 expression
increased both at early and late thaws (p<0.05), whereas
the adhesion marker L-selectin was decreased (p<0.05),
and endothelial marker CD31 was unchanged. EPC
viability decreased both at early and late time points
(p<0.1). There was no significant difference in markers
CD31, CD34, VEGFR2 and L-selectin in EPCs derived from
cryopreserved PBMC samples, but uptake of low-densitylipoprotein was increased after both 1 and 28 days of
cryopreservation (p<0.05). Adhesion and migration
properties of PBMCs and EPCs were unaffected by
cryopreservation.
Conclusion. Cryopreservation of PBMCs decreased
viability, but did not affect migrative or adhesive
functions. PBMCs were affected phenotypically, with
changes in CD34, VEGFR2 and L-selectin expression.
Overall, it appears that the more therapeutic EPCs
tolerate cryopreservation better than the heterogeneous
PBMC population.
Keywords. Endothelial Progenitor Cells, Cryopreservation,
Cell Viability
(10.O4) ESTABLISHMENT OF AN INDIVIDUAL HUMAN
VASCULAR CELL BANK CONSISTING OF UMBILICAL CORD
CELLS FOR THE TISSUE ENGINEERING OF VASCULAR
CONSTRUCTS
UNDER
GOOD
MANUFACTURING
PRACTICE (GMP) CONDITIONS
Polchow B (1), Hetzer R (1), Lüders C (1)
1. Department of Cardiothoracic and Vascular Surgery,
Laboratory for Tissue Engineering, Deutsches Herzzentrum
Berlin, Germany
Introduction. Fabricated tissue-engineered vascular
constructs could provide an alternative to conventional
vascular replacements. One of the bases for tissue
engineering of vascular constructs is an adequate cell
source. Cells from the human umbilical cord can be
directly isolated and cryopreserved until needed.
Currently no cell bank for human vascular cells is
available. Therefore, the establishment of a human
vascular cell bank conforming to GMP conditions,
including important quality controls such as cell viability,
cell growth and marker expression, is desirable.
Methods. A fundamental step was to adapt conventional
research and development agents to agents conforming
to GMP for the cell isolation, cultivation and
cryopreservation process. Vascular cells were isolated,
cryopreserved and recultured subsequently. Cell viability,
growth potential and the expression of cell-specific
markers from fresh and cryopreserved cells were studied
over several passages using Trypan blue staining, flow
cytometry analysis and immunofluorescence staining.
Results. Viability tests of directly thawed and recultured
cells demonstrated an increase of viability with rising
passage number and rapid adaptation to viabilities of
fresh cells. Growth potential of cryopreserved, recultured
cells was similar to that of fresh cultivated cells with
regard to the entire cultivation period. Furthermore, a
specific surface marker profile for vascular cells was
successfully established using FACS analysis. Fresh
cultivated and cryopreserved myofibroblasts were
positive for the cellular markers alpha- smooth muscle
actin, CD105, CD90, CD73, CD146 and HUVEC expressed
CD31, CD146, CD105 and CD144. Additionally
immunofluorescence staining using the same markers
was performed.
Conclusion. Adaptation of cell isolation, cell cultivation
and cryopreservation procedures to GMP conditions was
successful. For potential future applications standard
operating procedures (SOPs) and a validation process
have to be developed to make the establishment of an
individual human vascular cell bank feasible.
Keywords. cell bank, vascular umbilical cord cells, quality
controls, good manufacturing practice (GMP).
(10.O5) GROWTH ARREST OF HUMAN MSC HAS
DIFFERENT
EFFECTS
ON
OSTEOGENIC
AND
Dexheimer V (1), Janicki P (1), Richter W (1)
1. Orthopedic University Hospital Heidelberg, Center for
Experimental Orthopedics
Mesenchymal stem cells are a promising cell source for
tissue regeneration. During embryonal development
proliferation and differentiation are tightly linked because
proliferation is necessary in order to produce enough cells
for the differentiation step. We here asked whether
proliferation is an absolute requirement for successful
differentiation or if MSC can still differentiate after
growth arrest into osteogenic and chondrogenic lineage
in vitro.
Human MSC (n=5) were isolated from bone marrow and
expanded up to passage 3. In vitro chondrogenesis was
induced in a high density pellet culture system for 6
weeks and newly synthesized DNA in spheroids was
marked with BrdU at different time points. Additionally
spheroids were treated with Mitomycin C (20µm) before
and during differentiation. Success of differentiation
(proteoglycan-, collagen type II-deposition) and BrdUlabelling were detected histologically. Osteogenic
differentiation of treated and untreated MSC was induced
for three weeks. Mineral deposition and AlkalinePhosphatase activity were quantified. The BrdU-labelling
showed strong proliferation at day 1 of chondrogenic
induction which peaked again between day 14 and 21 in
areas becoming positive for collagen type II deposition.
Mitomycin blocked MSC proliferation while metabolic
activity was maintained.
Mitomycin-induced growth arrest of MSC before start of
induction or at distinct time points during the first 2
weeks
of
chondrogenic
induction
prevented
proteoglycan- and collagen type II-depostion according to
histology. Mitomycin treatment at later time points was
harmless. In contrast osteogenic parameters were
apparently not affected by growth arrest.
Proliferation in the early phase of differenation is a
requirement for successful chondrogenic differenation of
MSC in vitro but not for osteogenic differentiation.
Keywords. MSC, proliferation, differentiation
(10.P1) GLUCOSE REQUIREMENT FOR IN VITRO AN IN
VIVO SURVIVAL OF MESENCHYMAL STEM CELLS UPON
IMPLANTATION
Deschepper M (1), Oudina K (1), Manassero M (2),
Monfoulet L (1), Bensidhoum M (1), Logeart-Avramoglou
D (1), Petite H (1)
1. UMR 7052 CNRS ; 2. Ecole Veterinaire de Maisons
Alfort; Laboratoire de Recherches Orthopédiques (B2OA),
Faculté de médecine Lariboisière-Saint-Louis, Paris, France
The use of human mesenchymal stem cells (hMSCs) has
emerged as a potential new treatment of a variety of
diseases but has generated marginally successful results.
Actually, a consistent finding of most studies is the
massive death of transplanted cells. The underlying
reasons for the observed limited cell viability are not yet
fully understood but in vivo massive death of the
transplanted cells after engraftment into tissueconstructs is a major and serious problem. A possible
explanation for the aforementioned limited cell survival
upon implantation is that MSCs encounter an ischemic
(with low oxygen tension and nutrient depletion)
environment. In this study, we challenge the current
paradigm that gives a pivotal role to oxygen on hMSCs
massive cell death and hypothesize that exogenous
glucose and not only oxygen supply is required for
survival of hMSCs upon transplantation. To this aim,
hMSCs were exposed in vitro to sustain near anoxiahypoxic environment and the influence of exogenous
glucose on cell viability and functionality was assessed.
Results obtained showed that hMSCs were able to survive
21 days under sustained anoxia without serum providing
that they were cultured in the presence of glucose. These
results established that glucose depletion but not
sustained anoxia affected cell survival. Moreover, hMSCs
when cultured 21 days under anoxia in the presence of
glucose, kept their stemness and ability to differentiate
into osteoblasts and adipocytes. To further investigate
the role of glucose, MSCs were seeded onto scaffold
composites supplement or not with glucose and their
ability to enhance MSC survival was evaluated in an
ectopic mouse model. Results showed a striking increase
of cell viability in tissue construct supplement with
glucose. At day 14, a seven-fold increase in cell number
was observed in tissue constructs supplemented with
glucose when compared to the one of control tissue
constructs.
(10.P2) PLATELET-RICH CONCENTRATE IS PROTECTIVE
AGAINST
FLUOROQUINOLONE
INDUCED
EXTRACELLULAR MATRIX CHANGES IN HUMAN
TENOCYTES
Franklin SL (1), Zargar N (1), Poulsen RC (1), Willett K (1),
Thompson MS (1), Hulley PA (1)
1. University of Oxford
Introduction. Previous research has already been carried
out on the damaging effect fluoroquinalones have on
tendon cell viability (Zargar N. 2011). It has been shown
that platelet-rich concentrate (PRC) protects against this
cell death. The use of PRC as an adjunct in tendon repair
research is growing in popularity. The aim of this work is
to investigate the potential role of PRC in mitigating the
effects of fluoroquinolone therapy, notably Ciprofloxacin,
by studying extracellular matrix synthesis and
maintenance in a tendon cell culture model.
Methods. PRC was extracted from fresh human whole
blood via centrifugation, was immediately clotted and left
in medium overnight to release all biological factors.
Human tenocytes were treated over a 10 day period with
Ciprofloxacin with/out 10% PRC. The amount of collagen
and glycosaminoglycan’s (GAG) in the cell layer and
medium was measured using both Sircol™ soluble
collagen and dimethylmethylene blue assays respectively.
Results. Preliminary results show that from as early as 24
hours, up to at least 3 days, there is a significant
reduction of collagen in the cell layer in the presence of
Ciprofloxacin. This reduction is reversed by the addition
of 10% PRC. There are no significant changes in GAG
content in the cell layer, however the amount of GAG
released into the medium is reduced by Ciprofloxacin.
Addition of 10% PRC partially restored control levels.
Discussion and Conclusions. This study suggests that
Ciprofloxacin either directly or indirectly causes
disruption of the ECM, potentially explaining the
increased risk of Achilles rupture (Sode J. 2007). Addition
of PRC appears to reverse this effect. Detailed
mechanisms-of-action are unknown, therefore future
investigations will focus on gene expression changes of all
individual collagen types, decorin, and versican. This work
emphasises the potential for wider use of PRC in
protecting against environments damaging for tendon
cells and tissue.
Acknowledgements. This work was funded by Joint
Action.
Keywords. Tendon; Extracellular Matrix; Ciprofloxacin;
Platelet-rich concentrate
(10.P3) HGF-PRODUCING MESENCHYMAL STROMAL
CELLS SUPPORT CHRONIC LYMPHOCYTIC LEUKEMIC B
CELLS SURVIVAL
Giannoni P (1), Quarto R (2), Balleari E (3), Florio T (4),
Ferrini S (5), De Totero D (6)
1. Stem Cell Lab., Advanced Biotechnology Center,
Genova, Italy; 2. Dept. Experimental Medicine, University
of Genova, Genova, Italy; 3. Dept. Haematology, Hospital
San Martino, Genova, Italy; 4. Pharmacology Lab., Dept.
Oncology, Biology and Genetics, University of Genova,
Genova, Italy; 5. Immunological Therapies Lab., Natl. Inst.
for Cancer Research, Genova, Italy; 6. Gene Transfer Lab.,
Natl. Inst. for Cancer Research, Genova, Italy
Introduction.The longevity of chronic lymphocytic
leukemic B cells (CLL) in vivo contributes to leukemia
expansion and relapse. Cell survival capacity is lost in
vitro, evidencing the role of cellular interactions and
microenvironmental factors in CLL viability. Bone marrow
(BM) encompasses several cell types among which bone
marrow stromal cells (BMSC), capable to differentiate
along several lineages. Little is known on the effects of
undifferentiated/differentiated BMSC on CLL, nor which
BM-resident cells contribute to CLL progenitors
maintenance. We thus used BMSC and other cells of
mesenchymal origin to investigate the mechanisms of CLL
survival.
Methods. Co-cultures of mesenchymal-derived stromal
cells were performed with B cells from thirty leukemic
patients; CLL viability was assessed by annexin
V/propidium iodide flow cytometry analysis. Transwell
cultures and conditioned medium from the same cell
types were then tested to ascertain if viability could
depend upon released factors. Gene expression profiles
of differently supportive mesenchymal cells were used to
identify the soluble factors potentially involved in CLL
survival; signal-transduction and RNA interference assays
were then undertaken to verify this assumption.
Results. Co-cultures or conditioned medium of human
BMSC, osteoblasts-like MG63 cells or trabecular-bone
derived osteoblasts prolonged survival of CLL cells, while
chondrocytes or endothelial cells did not. Gene
expression analysis suggested a possible role of
hepatocyte growth factor (HGF) in CLL viability. Real-time
RT-PCR analysis demonstrated that HGF was produced
only by CLL-sustaining mesenchymal cells and that CLL
expressed c-MET, the HGF receptor. HGF addition to CLL
cultures enhanced CLL viability and induced Tyr705-STAT3
phosphorylation; both were inhibited by siRNA-mediated
HGF knockdown, as well as by inhibitors of STAT3
phosphorylation.
Conclusion. At the BM level, HGF contributes to apoptosis
resistance of CLL through the activation of c-MET/STAT3
axis. These results can be related to chronic lymphocytic
leukemia progression, suggesting new possible
therapeutic targets for the disease.
Keywords. chronic lymphocytic leukemia, mesenchymal
stem cells, growth factors, survival
Acknowledgements. Authors would like to acknowledge
CIBER-BBN, MAT2010-18155, CICYT- Spain and fellowship
CNPQ, Brazil.
Keywords. MSC; ceramic; cryogels
(10.P4) RESPONSE OF HUMAN OSTEOBLASTS AND
MESENCHYMAL STEM CELLS TO CRYOGELS BASED ON
THE
SYSTEM
2-(DIMETHYLAMINO)
ETHYL
METHACRYLATE / (2-HYDROXYETHYL) METHACRYLATE
αE/TRICALCIUM PHOSPHATE
Magalhaes, J (1), Burguera, EF (1), Blanci, FJ (1), Volkmer,
T (2), Sousa, V (3), Santos, L (3), Rodríguez-Lorenzo, L (4),
San Román, J (4)
1. INIBIC; 2. UNIFRA; 3. UFRGS; 4. ICTP
Introduction. Cryopolymerization is a clean processing
technique that produces highly hydrophilic and elastic
porous materials. The potential of this method in the
production of scaffolds for calcified tissue engineering has
been recently studied. The aim of this work is to study the
response of different cell types to the manufactured
cryogels.
Methods. Cryogels were prepared by free radical
copolymerization of the monomers (2-hydroxyethyl)
methacrylate (HEMA) and 2-(dimethylamino)ethyl
methacrylate (DMAEMA), α-tricalcium phosphate, and
N,N,N´,N´-tetramethylethylene diamine as activator, at 20ºC. Specimens with different monomer/water ratio (540), ceramic content (0-20%) and crosslinker
concentration (0-2%) were prepared. Biocompatibility
was tested with human osteoblasts and MSCs isolated
from bone marrow stroma, then expanded until 90%
confluent and cultured on the different cryogels for 7, 14
and 21 days. Cell viability was assessed, through the
alamar blue assay. Cell distribution and morphology were
determined by histological techniques. Expression of
type-I collagen and alkaline phosphatase (ALP) were
analyzed by immunohistochemistry.
Results. DMAEMA/HEMA ratios up to 25/75 were
studied. Greater porosity (75%) and pore size (1 mm) was
obtained for a 75/25 monomer ratio. 5% ceramic loaded
specimens produced an increase in the elastic modulus of
the specimens, from 1125 to 1161 Pa, for a 75/25
specimen while not affecting significantly the porosity of
the specimens. After 96h, both cell types had adhered
and proliferated on the materials’ surface (Fig.1). Material
colonisation could be observed along the 21 days of
culture, inferring their biocompatible profile. Expression
of type-I collagen could be detected whilst ALP expression
appeared to be correlated with α-TCP content.
As summary, results indicate that the materials tested are
biocompatible, showing vital cells adhering to the
materials, proliferating and giving evidence of early
expression of biochemical markers of osteoblastic
phenotype.
(10.P5) PHOTODYNAMIC RESPONSE OF PIGMENT CELLS
IN
ZEBRAFISH
LARVAE
INCUBATED
WITH
PHOTOSENSITIVE AGENTS
Álvarez MA (1), Ercolino JM (1)
1. Sección de Microscopía, Instituto Anatómico “José
Izquierdo”, Facultad de Medicina, Universidad Central de
Venezuela.
Introduction. In vitro experimental systems treated with
fluorescent organelle probes and photosensitizers, a
characteristic redistribution of fluorescence in cell
structure occurs after light irradiation. In vivo
experimental system the reversible changes in
pigmentation brought on by prolonged exposure to either
light or dark environments have reveled that this occurs
through relocalization of pigment organelles within cell,
changes in cell morphology and apoptosis. These
phenomena have promoted investigated on the etiology
of it, maybe mediated, among other way, through the
pigment cell itself.
Objective: The aim of this work was to examine the
photodynamic response of pigment cells in wild-type
zebrafish larvae from 3 dpf incubated with photosensitive
agents (PSs).
Material and Methods: Acridine orange 10-4 M,
methylene blue and toluidine blue 10-5 M were used.
Zebrafish larvae, from 3 dpf were partially immobilized
and placed on a LED array constructed for irradiation. It is
composed of a matrix of 4 light sources with emission
peak at 636 nm. The larvae were exposed during 5, 10
and 15 min. Embryo survival, melanophores cell
morphology with fluorescence microscopy was analyses
subcellular localization of PSs and statistical analysis.
Results: Photodynamic treatment resulted in a light dosedependent diminution of larvae survival. Pigmented
melanophores extend from the level of the hindbrain to
about the middle of the yolk ball. The cells showed the
typical stellate morphology of melanophores. After
irradiation the melanophores changes from initial stellate
appearance to punctuate form. Conspicuous changes in
the fluorescence pattern were observed.
Discussion: The photodynamic response of pigment cells
resulted in dramatic morphological changes probably
linked to a redistribution of melanosomes within cells or
changes in cell shape.
Keywords. relocalization of photosensitizers
Figure: Cells of the hatching gland present on the
pericardium over the anterior yolk sac revelated with
acridine orange.
(10.P6) STABILITY OF MESENCHYMAL STEM CELLS FROM
HUMAN CHIN BONE MARROW AFTER EXPANSION
PROCESS
Solarte VA (1), Arango ML (2), Franco LM (2), López JB (1),
Munera LM (2)
1. Universidad de Antioquia; Universidad Nacional de
Colombia; 2. Universidad de Antioquia
Mesenchymal stem cells (MSCs) offer great therapeutic
potential in regenerative medicine because of its
features: readily available from several sources, high in
vitro proliferation, immunomodulating capacity and injury
site migration among others. However, due to their low
percentage within different sources (0.1 - 0.0001%), it is
necessary to subject them to expansion processes that
can lead to genomic instability, cellular dysfunction and
malignancies development after transplantation. Human
chin bone marrow is a potential new source for MSCs
isolation, however, expansion for an extended period is
required, and is necessary to assess their stability after
this process. In this study we analyzed morphological
changes and nuclear chromatin by cytogenetic and comet
assay of human chin bone MSCs samples expanded for
approximately 11 generations. Preliminary results did not
show chromosomal instability neither significant
clastogenic effect (absence of single strand breaks in
DNA) in analyzed samples. Nevertheless, morphological
changes, increased population doubling time, decreased
ability to form colonies, differential condensation of
nuclear chromatin in interphase nuclei and nuclear
malformations after expanding by 11 generations were
observed in three samples. Although preliminary studies
showed that MSCs isolated from chin can be expanded,
further studies are necessary to design and to ensure safe
cell therapies.
Keywords. Cellular therapies, Cell stability, Chromosomal
abnormalities
(10.P7) STATUS OF CELL TRANSPLANTATION IN IRAN
Aghayan HR (1), Arjmand B (1), Larijani B (1), Manavi SM
(2)
1. Endocrinology and Metabolism Research Center,
Tehran University of Medical Sciences, Tehran, Iran; 2.
Iran Presidency Technology Cooperation Office
In recent years, like many other countries, modern cell
therapy has been started in Iran. Autologous Schwann cell
transplantation for spinal cord injury, Mesenchymal stem
cell transplantation for multiple sclerosis, cirrhosis,
diabetes
and
myocardial
regeneration,
and
Hematopoietic fetal stem cell transplantation for
diabetes, cirrhosis and multiple sclerosis are prominent
examples of current clinical trials. These trials are
generally regulated by scientific and ethical committees
of medical universities but two of them have been
registered in ministry of health (as national project). Lack
of national regulation and defined standards is the main
safety concern for cell therapy projects and each cell bank
has its own quality policy and referred standards (like
cGMP, cGTP and GLP). Recently Food and Drug
Organization (Ministry of Health) has started working on a
plan to regulate and harmonize cell and tissue banking
activity in Iran. In the end of 2010 the draft of national
standard for cell and tissue banking was announced that
can be a basic reference for cell therapy center as a
minimal safety standard. In order to achieve the higher
level of safety the authors recommend that a more
specialized national standard for cell therapy should be
publishing by the government. We also believe that
implementation of some general quality management
system based on the ISO 9001 and ISO13485 can improve
safety of cell based products.
Keywords. Cell therapy, Iran, ISO, GMP
(10.P8)
CO-CULTURE
OF
OSTEOBLASTIC
AND
ENDOTHELIAL
CELLS
IN
THE
PRESENCE
OF
BISPHOSPHONATES
Ribeiro V (1), Garcia M (1), Oliveira P (2), Pires MJ (2),
Colaço B (2), Fernandes MH (1).
1. Universidade do Porto, Faculdade de Medicina Dentária
(FMDUP),
Laboratório
de
Farmacologia
e
Biocompatibilidade Celular, Portugal; 2. CECAV.
Departamento de Ciências Veterinárias. Universidade de
Trás-os-Montes e Alto Douro, Portugal.
Introduction. Long-term therapies with Bisphosphonates
(BPs) can cause osteonecrosis of the jaws, a condition
characterized by tissue dehiscence, chronic bone
devitalization, hypocellularity and lytic radiographic
features, being usually refractory to therapy. Among the
diverse and complex mechanisms of action recently
suggested for BPs, the anti-angiogenic properties appears
to be a relevant feature of their pharmacological profile.
Due to the intimate relationship between angiogenesis
and osteogenesis during bone formation events, the aim
of this study was to analyze the effect of representative
BPs – alendronate and zoledronate, two widely used BPs
in therapeutics - in the behavior of a co-culture system of
human osteoblastic and endothelial cells.
Methods. MG63 osteoblast-like cells (103 cell/cm2) and
human dermal microvascular endothelial cells (104
cell/cm2) were cultured isolated or co-cultured in
endothelium medium, in the presence of 10-12 to 10-6 M
Alendronate or Zolendronate. Cultures were maintained
for
14
days
and
characterized
for
cell
viability/proliferation (MTT assay), pattern of cell growth
(CLSM) and gene expression of osteoblastic and
endothelial markers (RT-PCR; Collagen type1, ALP, BMP-2,
OPG, M-CSF, CD31, VE-Cadherin and vWF).
Results. In control conditions (absence of BPs), cocultures of osteoblast and endothelial cells maintained
the viability/proliferation and presented a characteristic
pattern of cell growth, i.e. the formation of cell clusters of
endothelial cells surrounded by osteoblast cells, and
inductium and/or earlier expression of osteoblastic and
endothelial markers. The presence of Alendronate or
Zolendronate did not affect cell viability/proliferation but
caused decreased gene expression of endothelial
associated markers in monocultures and co-cultures.
Conclusion. The inhibitory effects of BPs in endothelial
cells might play a role in the deleterious effects of BPs in
the bone tissue.
References:
1. Wood J, Bonjean K, Ruetz S, et al. The Journal of
Pharmacology and Experimental Therapeutics 2002; 302:
1055-1061.
2. Carano, R.A.D., Filvaroff, E.H. Drug Discovery Today
2003; 21:980-989.
Keywords. Bisphosphonates, osteoblast cells, endothelial
cells, co-culture
(10.P9) ISOLECTIN OF PHYTOHEMAGGLUTININ-INDUCED
APOPTOTIC PATHWAY IN LUNG CANCER CELLS
Kuo WT (1), Yao CH (2), Lin FH (3)
1. Ph.D. Program in Tissue Engineering and Regenerative
Medicine, National Chung Hsing University, Taichung,
Taiwan; 2. Department of Biomedical Imaging and
Radiological Science, China Medical University, Taichung,
Taiwan; 3 . Division of Medical Engineering Research,
National Health Research Institutes, Miaoli, Taiwan
Apoptosis is a physiological mechanism required for
maintaining cell numbers and removing unnecessary cells.
Deregulation of apoptosis will result in many diseases
including cancer. Lung cancer is the leading cause of
cancer-related death all over the world. In prior research
reports of cancer therapy, phytohemagglutinin (PHA), the
lectin extracted from red kidney beans, demonstrated the
ability to inhibit the growth of human cancer cells.
However, one of its isoforms, erythroagglutinating (PHAE) has yet to be evaluated on its anti-cancer effects
against lung cancer cells A-549.
First, we used MTT assay and G6PD release assay to
evaluate cell viability and cytotoxicity on A-549 cells.
Next, PHA-E was used to induce apoptosis in order to
determine the possible signal transduction pathway, as
measured by flow cytometry assays, fluorescent stains
and western blot analysis.
The results showed that PHA-E treatment caused a dosedependent increase of cell growth inhibition and
cytotoxicity on A-549 cells. In Annexin V/PI and TUNEL/PI
assay, we found that the rate of apoptotic cells was raised
as the concentration of PHA-E increased. In addition, cell
morphological changes, chromatin condensation and
fragmentation, were observed by DAPI/TUNEL stain after
treatment with PHA-E. Treatment of A-549 cells with
PHA-E resulted in enhancing the release of cytochrome c,
which thus activated an increase in protein levels of
caspase-9 and caspase-3, up-regulation of Bax and Bad,
down-regulation of Bcl-2 and phosphorylated Bad, and
finally the inhibition of epidermal growth factor receptor
and its downstream signal pathway PI3K/Akt and
MEK/ERK.
In conclusion, PHA-E can induce growth inhibition and
cytotoxicity of lung cancer cells, which is mediated
through activation of the mitochondria apoptosis
pathway. These results suggest that PHA-E can be
developed into a new therapeutic treatment that can be
applied as an effective anti-lung cancer drug in the near
future.
Keywords. Apoptosis; Lung cancer; Phytohemagglutinin
Erythroagglutinating
(10.P10) CORTICOIDS ALLEVIATE RSV-INDUCED LOSS OF
CILIATED CELLS AND ENHANCED MUC5AC IN
DIFFERENTIATED HUMAN BRONCHIAL EPITHELIAL CELLS
(D-HBE)
Mata M (1), Cortijo J (2), Banyuls P (3), Armengot M (4),
Carda C (5)
1. Fundación de Investigación del Hospital General
Universitario de Valencia; CIBERES; Facultad de Medicina,
Departamento de Patología; 2. Fundación de
Investigación del Hospital General Universitario de
Valencia; CIBERES; Facultad de Medicina, Departamento
de Farmacologia; 3. Universidad de Valencia, Facultad de
Medicina, Departamento de Farmacología; 4. Fundación
de Investigación del Hospital General Universitario de
Valencia; Universidad de Valencia, Facultad de Medicina,
Departamento de Cirugía; 5. Universidad de Valencia,
Facultad de Medicina, Departamento de Patología.
INCLIVA; CIBER-BBN
Introduction: Respiratory syncytial virus (RSV) may cause
COPD exacerbations. Anti-inflammatory compounds
reduce the risk of COPD exacerbations in clinical studies.
This study investigated whether in vitro dexamethasone
influences the interaction of RSV with ciliated D-HBE.
Methods: D-HBE from standard air-liquid interface
culture were infected with RSV (MOI 0.3 PFU/cell) in the
presence of dexamethasone 1 µM or vehicle. After 10
days the following was measured (i) number of ciliated
cells based on assessment of cilia activity by high-speed
video microscopy, (ii) cilia markers FOXJ1 and DNAI2
(mRNA), (iii) MUCAC (mRNA) and (iv) ultra-structure of
cilia by electron microscopy. Results are given as mean ±
SEM from 10 (number of ciliated cells), 3 (mRNA analyses)
and 1 (TEM) independent experiments.
Results: At day 10 following RSV infection the number of
ciliated cells declined from 300 ± 5 / field to 68.2 ± 20.5 /
field. This was partially prevented by dexamethasone
(194.8 ± 14.7 / field; p<0.05 vs RSV). His results are in line
with TEM analysis of cilia ultrastructure. In line, RSV
reduced FOXJ1 and DNAI2 transcripts to 53.7 ± 0.1 % and
19.2 ± 0.2 % of control, respectively. Dexamethasone 1
µM fully prevented the loss in DNAI2 and partially
restored expression of FOXJ1 to 74.5 ± 0.2 % of control
(p<0.05 vs RSV). In parallel, a 4.87 ± 0.3-fold rise in
MUC5AC mRNA secondary to RSV was abolished in the
additional presence of dexamethasone.
Conclusion: In differentiated human bronchial epithelial
cells RSV caused a loss of ciliated cells and associated
markers while MUC5AC expression was increased.
Dexamethasone 1 µM reversed these effects.
Acknowledgements: This work is supported by grants of
the local government of Valencia (Conselleria de Sanitat),
the Spanish Ministery of Science and innovation
(SAF2008-03113) and the Health Institute Carlos III
(CIBERES, CB06/06/0027).
Keywords. Cilia, airway epithelial cells, ALI, COPD,
tobacco smoke, corticoids, inflammation
(10.P11) PLATELET RICH PLASMA PROTECTS TENOCYTES
FROM DRUG-INDUCED SENESCENCE AND DEATH
Zargar-Baboldashti N (1), Poulsen RC (1), Franklin SL (1),
Thompson MS (1), Hulley PA (1)
Introduction. Tendon disorders are frequent and cause
month-long disabilities due to poor healing mechanisms.
The underlying causes of tendon diseases are not fully
understood and effective treatments are limited.
Certain drugs such as dexamethasone and ciprofloxacin
interfere with innate healing processes and are thought
to predispose tendons to rupture, presenting clinically
relevant tools with which to investigate damage
mechanisms in tendon. However, both drugs are highly
effective in treatment of inflammatory and infectious
conditions, therefore new strategies to minimize their
adverse effects are of strong interest. Platelet rich plasma
(PRP), a rich autologous source of growth factors, has
been used to enhance tendon healing. This study
investigated the effects of both drugs on parameters of
human tenocyte viability, senescence and death.
Secondly, the possible use of PRP to mitigate negative
effects of both drugs was tested.
Materials and Methods. Centrifuged PRP, from fresh
human whole blood, was immediately clotted and left in
medium overnight to release biological factors. Human
hamstring tenocytes were exposed to ciprofloxacin and
dexamethasone with / without PRP. Alamar Blue, βgalactosidase assay and live / dead stain were used to
measure respectively viability, senescence and death in
tenocytes.
Results. The viability of tenocytes treated with
ciprofloxacin decreased dose-dependently, with no
induced senescence but increased cell death.
Dexamethasone reduced viable cell number without
overt cell death but the number of senescent cells
increased up to 50%. After co-treatment with 10% PRP
viable cell number increased significantly in both
conditions and dexamethasone-induced senescence was
reduced to 8%.
Conclusion. We demonstrated that ciprofloxacin and
dexamethasone have differing adverse effects on human
tenocytes, ciprofloxacin inducing cell death while
dexamethasone primarily induces senescence. Since it is
necessary to continue using dexamethasone and
ciprofloxacin therapeutically, our results suggest that coinjection of PRP could block side-effects of these drugs
and promote healing in tendons.
Keywords. Platelet Rich Plasma, Dexamethasone,
Ciprofloxacin, tenocyte
(10.P12) EX VIVO CYTOTOXIC EFFECTS OF ANTIGLAUCOMA PROSTAGLANDIN ANALOGUES ON HUMAN
CONJUNCTIVAL CELLS
Pérez-Roca F (1), Rodrigo-Morales E (1), Ramos JF (1),
González-Andrades M (2,3), Garzón I (2), Oliveira ACX (2),
Alaminos M (2)
1. Division of Ophthalmology, University Hospital Virgen
de las Nieves, Granada, Spain; 2. Tissue Engineering
Group, Dept. Histology, University of Granada, Spain; 3.
Division of Ophthalmology, University Hospital San Cecilio,
Granada, Spain
Background. Glaucoma is a leading cause of blindness in
the world. Elevated intraocular pressure is the most
important risk factor in its pathogenesis and most of the
clinicians choose medical therapy with prostaglandin
analogues (PGs) as first option of treatment. However,
cytotoxic effects of these drugs on the human conjunctiva
are not well known. In this work, we have evaluated the
cytotoxic effects of several PGs ophthalmic solutions
using an ex vivo cell culture model.
Methods. Primary cell cultures of human conjunctival
fibroblasts were stablished from biopsies of healthy
patients. The cells were isolated by enzymatic digestion.
These cells were maintained using Dulbecco's Modified
Eagle Medium (DMEM) supplemented with 10% fetal
bovine serum. We investigated the cytotoxic effects of
sequential dilutions bimatoprost, tafluprost, travoprost
and latanoprost on human conjunctival cell cultures by
using WST-1 method, a non-radioactive colorimetric
quantitative assay that measures mitochondrial enzyme
activity, which is directly proportional to the number of
viable cells. We evaluated each PGs for 5, 30 and 60
minutes at 6 different concentrations.
Results. The WST-1 assay suggested that the four PGs
showed a significantly higher level of cytotoxicity in
higher concetrations. Tafluprost showed a less toxic
profile at the three times of exposition while the
latanoprost seems to be most harmful, although this
differences decrease when the drug was pure.
Conclusions. The WST-1 is a reliable technique for
assessing citotoxity in conjunctival cells. In our study, all
drugs showed significant levels of cytotoxicity at higher
concentrations, although tafluprost seems to be less toxic
at these times. This fact could likely be related to the fact
that tafluprost does not have benzalkonium chloride on
solution. Further studies are needed to clarify the role of
this preservative in cell death.
(10.P13) CELL VIABILITY QUALITY CONTROL OF DENTAL
PULP STEM CELLS FOR TISSUE ENGINEERING
Martín-Piedra MA (1), Oliveira ACX (1), Garzón I (1),
Rodríguez IA (1), Alfonso C (1), Sánchez-Quevedo MC (1),
Campos A (1)
1. Tissue Engineering Group, Dept. Histology, University of
Granada, Spain
Introduction. Dental pulp stem cells (DPSCs) have been
recently reported as a potential reservoir of cells with
high differentiation and transdifferentiation capabilities
that allows the construction of new tissues in regards to
the therapeutical needings. In this context, the
identification of DPSCs´s cell viability profile could be
promising for tissue engineering protocols. The aim of
this study was to determine the viability patterns of
(DPSC) in order to establish the ideal passage for the use
in Tissue Engineering.
Methods. Dental pulp stem cells (DPSCs) were isolated
from two extracted human third molars by enzymatic
digestion. Primary cell cultures were maintained under
standard cell culture conditions and trypsinized along 10
sequential passages. Firstly, for cell viability study trypan
blue and LIVE/DEAD® assays were performed at the 10
passages. Secondly, WST-1 cell proliferation assay were
also carried out in all study samples. Statistical analysis
was done by U Mann-Whitney and Kruskal Wallis test.
Results. DPSCs were viable > 85% during all the study
although there were differences on cell viability, detected
by each of the assays carried out, between different
DPSCs subcultures. From primoculture to fourth passage,
viability results were wavering due to an adaptation stage
to the in vitro conditions. At fifth passage viability
increases from 91,27% to 97,53% (p = 0,0003),
maintaining an slight upward trend to eighth passage:
96,72% at sixth passage (p = 0,798). At seventh
subculture, viability showed an slight decrease to 95,03%
(p = 0, 020) that was offset at the next passage, reaching
the top-point of viability: 96,97% (p = 0,015). From here,
viability seems to keep constant until the end of the
study.
Conclusions. After four subcultures cells were adapted to
in vitro environment. DPSCs at eighth passage showed the
highest viability, suggesting the ideal conditions for use in
Tissue Engineering.
11. CELL-BASED THERAPIES AT BEDSIDE
Chair: Dimitrios I. Zeugolis
Co-chair: Yury Rochev
Keynote speaker: Masayuki Yamato
Organizer: Dimitrios I. Zeugolis
Synopsis: Injuries and degenerative diseases constitute a
bottleneck in medical and surgical practice. As the human
population ages and life expectancy increases, injuries
and degenerative conditions will continue to rise putting
a financial strain on healthcare. It is therefore imperative
to develop functional tissue regeneration strategies.
Natural or synthetic scaffold-based therapeutic
approaches are traditionally used to improve
regeneration and functional recovery. However,
advancements in molecular and cell biology have allowed
the use of cell-based therapies for tissue engineering and
regenerative medicine applications. The driven
hypothesis of this venerable concept is that replacement,
repair and restoration of function can be accomplished
best using cells that will create their own host-specific
extracellular matrix. Indeed, cells are professional matrix
makers and assemble into large aggregates together with
ligands, growth factors and other matrix components
with a precision and stoichiometric efficiency that is still
unmatched by man-made devices, recombinant
technologies derived components or chemical
compounds. Cell-based injectable systems and cell-sheets
derived from autologous primary cell isolates; from
established cell lines; and from a variety of stem cells
have been used for numerous clinical targets, including
cornea, skin, blood vessel, cartilage, lung, cardiac patch,
oesophagus and periodontal applications. This
symposium aims to highlight clinical applications of
scaffold-free cell-based therapies and discuss key
advancements and current hurdles that still prohibit the
widely adaptation of this technology in Tissue Engineering
and Regenerative Medicine.
(11.KP) CELL SHEET ENGINEERING FOR REGENERATIVE
MEDICINE: ITS CURRENT STATUS OF CLINICAL
APPLICATIONS AND SUPPORTING TECHNOLOGIES
Yamato M (1)
1. Institute of Advanced Biomedical Engineering and
Science, Tokyo Women's Medical University
We have developed a novel strategy for regenerative
medicine to recover tissue functions by using
temperature-responsive cell culture surfaces on which
temperature-responsive polymer is covalently grafted by
electron beam irradiation or other chemical reactions.
These surfaces achieve temperature-responsive cell
adhesion and detachment with no need for proteolytic
enzyme such as trypsin and dispase. To overcome the
limits of conventional tissue engineering methods such as
the use of single-cell suspension injection and the use of
biodegradable polymer scaffolds, we have applied
transplantable cell sheets fabricated with temperatureresponsive culture surfaces for cell delivery. Only by
reducing temperature around room temperature, all the
cells are harvested from the dish as a single contiguous
cell sheet. Since these cell sheets retain extracellular
matrix deposited during culture below them, integration
to tissue or other cell sheets is observed immediately
after the transplantation. Here, we show the pipelines
and current status of clinical applications of regenerative
medicine using cell sheet engineering. Skin and corneal
defects have been treated with transplantable cell sheets
fabricated on the surfaces. In bilateral cases, patients’
own oral mucosal epithelial cells are utilized as the cell
source, since both eyes are damaged and no epithelial
stem cells are obtained from the patients. Now, we have
performed the clinical trial under EMEA (European
Medicines Agency) of the corneal regenerative medicine
in Europe. We expect that we will obtain the approval in
2011. Severe heart failure was also treated with cell
sheets fabricated from patient’s own skeletal myoblasts.
Esophageal defects after endoscopic tumor dissection
have been treated by cell sheet engineering. In these
cases, we also utilize patients’ own oral mucosal epithelial
cells as the cell source. We expect further improvements
of stimuli-responsive culture surfaces will realize the
reconstruction of more complex tissues to potentially
treat a wide range of diseases.
(11.O1) ENDOTHELIAL CELLS POTENTIATE CELL SHEETS
OSTEOGENIC ABILITY
Pirraco RP (1,2), Iwata T (1), Marques AP (2), Yamato M
(1), Reis RL (2), Okano T (1)
1. ABMES, Tokyo Women’s Medical University, Tokyo,
Japan; 2. 3B's Research Group, University of Minho,
Guimarães, Portugal
Introduction. Bone Tissue Engineering strategies based
on the use of scaffolds and osteogenic cells present
drawbacks such as cell necrosis at the bulk of the scaffold
related to poor vascularization of the constructs. Cell
sheet (CS) engineering has been proposed as a successful
scaffold-free alternative for the regeneration of several
tissues. The use of this technology is herein proposed for
bone regeneration by combining osteogenic CSs and
endothelial cells.
Materials and Methods. Osteogenic CSs were created by
differentiating male rat bone marrow cells (rBMSC) in
thermo-responsive culture dishes in osteogenic medium.
Human umbilical vein endothelial cells (HUVECs) were
seeded on the rBMSCs to create co-cultured CSs. The CSs
were recovered by lowering the temperature; the
osteogenic CSs were stacked on top of either a cocultured or a similar osteogenic CS and transplanted to
female nude mice. Implants were recovered after 7 days
and characterized by hematoxilin&eosin (H&E) and
alizarin red (AR) stainings, immunohistochemistry for
osterix, osteopontin, SRY (to identify transplanted male
rat cells) and CD31, and calcium quantification.
Results. H&E and AR stainings showed mineralized tissue
formation in the implants both with and without HUVECs.
Osterix and SRY immunostaining demonstrated the
presence of host and donor osteogenic cells at the
mineralization site. HUVECs contribution to neovascularization was confirmed by human CD31
identification. Furthermore, calcium quantification results
(figure 1) showed a higher degree of mineralized tissue
after the transplantation of the constructs with HUVECs.
Conclusions. This work confirmed the potential of
transplanted osteogenic cell sheets for bone regeneration
as well as the advantage of promoting cross-talk between
osteogenic and endothelial cells for improved new tissue
formation. The proposed approach avoids the constraints
of scaffold use while successfully addressing the
important issue of implant vascularization.
Acknowledgements. PhD grant SFRH/BD/44893/2008 to
R.P. Pirraco by the Portuguese Foundation for Science
and Technology is acknowledged.
Keywords. Cell sheet engineering, tissue engineering,
bone, endothelial cells
(11.O2)
MODULATION
OF
THE
IN
VITRO
MICROENVIRONMENT
USING
MACROMOLECULAR
CROWDING
Satyam A (1), Joshi L (2), Raghunath M (3), Pandit A (1),
Zeugolis D (1)
National University of Ireland Galway, Ireland; 2. National
Centre for Biomedical Engineering Science, National
University of Ireland Galway, Ireland; 3. Tissue
Modulation Laboratory, Division of Bioengineering,
Faculty of Engineering, National University of Singapore,
Singapore
Introduction. In vitro, cells are customarily cultured in
highly diluted aqueous conditions which are disgustingly
disparate from the macromolecularly crowded
microenvironment they have been derived from. As a
consequence, cells lose in vitro their phenotype,
functionality and therapeutic potential. Recent reports
show that macromolecular crowding (MMC) - the
addition of macromolecules to culture media, not only
enhances the deposition of extracellular matrix, but also
preserves cell phenotype. Here, we analysed the
influence of various crowding molecules on in vitro
deposition of extracellular matrix from human lung and
skin fibroblasts under variable serum concentrations.
Methods. Human primary fibroblasts (e.g. lung, skin)
were cultured under MMC (e.g. 100µg/ml dextran
sulphate; 37.5mg/ml Ficoll™70 and 25mg/ml Ficoll™400;
and 100 µl/ml sepharose-CL) and various serum
concentrations (0.0 to 10%). The influence of various
crowders on cell morphology and metabolic activity was
evaluated using phase-contrast microscopy and
alamarBlue® assay respectively at day 2, 4 and 6. The
deposition of extracellular matrix proteins was analysed
by SDS-PAGE and immunocytochemistry for collagen
type-I and fibronectin.
Results. Phase-contrast microscopy (Figure-1A) revealed
that the fibroblasts maintained their spindle-shaped
morphology independent of macromolecular crowder
present or the serum concentration up to 6-days in
culture. AlamarBlue® analysis demonstrated that cell
metabolic activity was not affected independent of the
macromolecular crowder present or the serum
concentration even up to 6-days in culture (p>0.05) (not
shown). Densitometric analysis (Figure-1B) of SDS-PAGE
demonstrated that MMC significantly increase collagen-I
deposition (p<0.0001) at all tested serum concentrations.
Immunocytochemistry (Figure-1C) further confirmed the
enhanced deposition of collagen-I and its co-localisation
with fibronectin in presence of macromolecular crowders.
Conclusions.
Modulation
of
the
in
vitro
microenvironment with macromolecular crowding not
only maintains cell-viability and morphology, but also
enhances extracellular matrix deposition even under low
or even zero serum supplementation.
Acknowledgments. Science Foundation Ireland (Grant09/RFP/ENM2483) and SFI-ETS-Walton award for
financial support.
Keywords. Macromolecular Crowding, Collagen Type I
Deposition, Human Skin and Lung Fibroblasts, Serum
Concentration
and functional efficacy in cardiac ischemia, 70 nude rats
underwent myocardial infarction. Two weeks later,
animals received at the infarction border either 107 cells
of one of the 3 treatment groups or PBS. Four weeks
post-treatment, the ejection fraction was significantly
worsened by treatment with either ALL VEGF (-13.4%) or
control (CD8) cells (-8.8%) as well as the PBS group (8.0%) compared to SPEC VEGF cells (+1.7%). Initial
histology results confirm the induction of aberrant
structures in the ALL group, which were completely
prevented by SPEC cells similarly to the non-ischemic
tissue.
Conclusions. Controlled VEGF delivery by FACS-purified
ASC is effective to reliably induce only normal vascular
growth in the myocardium and is a promising novel
strategy to achieve safe and therapeutic angiogenesis to
treat cardiac ischemia.
Keywords. Angiogenesis, FACS, myocardium, VEGF
Figure 1A: Phase-contrast microscopy of fibroblasts under
crowded (with dextran suphate) and non-crowded
conditions at different FBS concentration. Figure 1B:
Densitometric analysis of SDS-PAGE gels for collagen I
deposition Figure 1C: Immunocytochemistry for Collagen
I (green) and Fibronectin (red). Nuclei were
counterstained with DAPI (blue).
(11.O3) CONTROLLED VEGF EXPRESSION ENSURES SAFE
ANGIOGENESIS AND FUNCTIONAL IMPROVEMENT IN A
MODEL OF MYOCARDIAL INFARCTION
Melly L (1,2), Marsano A (1), Helmirch U (1), Heberer M
(1), Eckstein F (2), Carrel T (3), Cook S (1,3), Giraud-Flück
MN (1,3), Tevaearai H (1,3), Banfi A (1,3)
1. Cell and Gene Therapy, Basel University Hospital; 2.
Cardiac Surgery, Basel University Hospital; 3. Department
of Cardiovascular Surgery, Inselspital, Bern University
Hospital
Introduction. VEGF can induce normal or aberrant
angiogenesis depending exclusively on the amount
secreted in the microenvironment. To make this concept
clinically applicable, we developed a FACS-based
technique to rapidly purify transduced progenitors that
homogeneously express a specific VEGF level from a
heterogeneous primary population. Here we aim at
inducing safe and efficient angiogenesis in the heart by
cell-based expression of controlled VEGF levels.
Method and Results. Human adipose-tissue stem cells
(ASC) were transduced with retroviral vectors expressing
either rat VEGF linked to the FACS-quantifiable surface
marker CD8, or CD8 alone (CD8) as control. VEGFexpressing cells were then FACS-purified to generate
populations producing either a specific (SPEC) or
heterogeneous (ALL) VEGF levels. In a non-ischemic study,
107 cells of each treatment group (CD8, SPEC, ALL) were
injected into the myocardium of nude rats. After 4 weeks,
vessel density was increased 2-3 fold by both VEGFproducing groups. However, ALL cells caused the
development of numerous aberrant angioma-like
structures, while SPEC cells induced only normal and
stable angiogenesis (Figure 1). To determine the safety
(11.O4) HUMAN UMBILICAL CORD PERIVASCULAR STEM
CELLS (HUCPVCS) AND THEIR CONDITIONED MEDIA
INCREASE
PROLIFERATION,
SURVIVAL
AND
DIFFERENTIATION IN THE DENTATE GYRUS OF ADULT
RAT HIPPOCAMPUS
Teixeira FG (1), Carvalho MM (1), Silva NA (2), Neves NM
(2), Reis RL (2), Sousa N (1), Pinto L (1), Salgado AJ (1)
1. Life and Health Sciences Research Institute, School of
Health Sciences, University of Minho, Braga, Portugal; 2.
3B's Research Group - Biomaterials, Biodegradables and
Biomimetics, University of Minho, Guimarães, Portugal
Recently, it was shown in vitro that the conditioned
media (CM) of human umbilical cord perivascular cells
(HUCPVCs) are able to modulate the survival, viability and
proliferation of neural precursors, neurons and glial cells.
However, in vivo studies, particularly in the brain regions
where neurogenesis occurs, were still missing. Therefore,
the main aim of this work was to analyse the effect of
HUCPVCs and their CM on the proliferation, survival and
differentiation of dentate gyrus (DG) resident cells in the
adult rat hippocampus. HUCPVCs were isolated from the
perivascular region of the human umbilical cord and their
CM was collected 24 hours after conditioning. Animals
were sacrificed 1, 4 and 12 weeks after injections of
HUCPVCs or their CM in the DG for immunohistochemical
characterization of the above referred parameters.
Results revealed that the effects of HUCPVCs and their
CM in the DG resident cells had different trends.
Concerning the animals injected with HUCPVCs, we
observed an increase on the proliferation (Ki-67 and BrdU
positive cells) both at one week and one month after.
Moreover in this group it was also possible to observe
that a small percentage of HUCPVCs were co-localizing
with GFAP, which indicates a possible differentiation of
these cells towards astrocytes. On the other hand, in the
animals injected with CM, the effect caused in the DG was
more evident for astrocytes (GFAP+) and neuronal (MAP2+) cell densities. Finally, the CM were also able to induce
the differentiation of resident neural precursor towards
the neuronal and astrocytic lineages. With this work it
was possible to show, for the first time, that the HUCPVCs
secretome is able to modulate the in vivo induction of
cellular proliferation, survival and differentiation. This
observation opens up a good perspective for the
application of HUCPVCs and their CM in regenerative
medicine approach.
Keywords. Secretome, Umbilical cord, Stem cells, Neural
differentiation
(11.P1) AXON FORMATION IN THE EMBRYONIC STEM
CELL-DERIVED MOTONEURON
Shen CI (1), Su HL (2)
1. Department of Veterinary Medicine, National ChungHsing University; 2. Department of Life Sciences, National
Chung-Hsing Univeristy
Developing neural cell must form a highly organized
architecture to properly receive and transmit nerve
signals. Neural formation from embryonic stem (ES) cells
provides a novel system for studying axonogenesis, which
are orchestrated by polarity-regulating molecules. Here
the ES-derived motoneurons, identified by HB9 promoterdriven green fluorescent protein (GFP) expression,
showed characteristics of motoneuron-specific gene
expression. In the majority of motoneurons, one of the
bilateral neurites developed into an axon that featured
with axonal markers, including Tau1, vesicle acetylcholine
transporter and synaptophysin. Interestingly, one-third of
the motoneurons developed bi-axonal processes but no
multiple axonal GFP cell was found. The neuronal
polarity-regulating proteins, including the phosphorylated
AKT and ERK, were compartmentalized into both of the
bilateral axonal tips. Importantly, this aberrant axon
morphology was still present after the engraftment of
GFP⁺ neurons into the spinal cord, suggesting that even a
mature neural environment fails to provide a proper
niche to guide normal axon formation. These findings
underscore the necessity for evaluating the
morphogenesis and functionality of neurons before the
clinical trials using ES or somatic stem cells.
Keywords. Motoneuron, embryonic stem cells
12. CHARACTERIZATION OF TISSUE
MECHANICS
Chair: Guillermo Rus
Keynote speaker: Quentin Grimal
Organizer: Guillermo Rus
Synopsis: The rational principles of solid mechanics are
an exciting framework to understand, monitor and
control functional tissue engineering and quality at all
scales form cell to organ. Tissue mechanics understanding
can also be extended to diagnose pathologies that
manifest by tissue consistency changes, such as
pulmonary and coronary arterial walls, tumours or
osteoporosis, just to mention a few, but also for
therapeutic uses: for instance, as a means to alter the
pharmacokinetics and drug permeability through cell
membranes, ranging from transdermal drug delivery to
gene therapy. Addressing tissue biomechanics requires a
concerted, collaborative effort between engineers,
physicists and clinicians.
A particularly active research area is currently growing on
exploring various physical principles to quantify
mechanical properties of tissue. They can be classified as
invasive, like indentation, tensile or compression testing,
or non-invasive and on-line technologies, like ultrasound,
high-frequency ultrasound, vibroacoustography, X-ray or
MRI-based elastography, among others. Understanding
the complex mechanical laws that govern soft tissues is
also a fundamental challenge. They show non-linear,
hysteretic, viscoelastic and in some cases also viscoplastic
behaviour, in addition to following a heterogeneous and
anisotropic pattern.
This symposium covers the multiple disciplines that the
characterization of tissue mechanics requires:
mathematical models, reconstruction algorithms, inverse
problems, sensor engineering, physiology, histology or
biochemistry, just to begin the list. The scientific
challenge relies on searching expertise and control
ranging all the way from the ground research on the
challenging physics interaction with tissue to the applied
development of tissue engineered materials, and
understanding in-depth from the micromechanical scale
of the tissue to the organ-level physics.
(12.KP)
ULTRASONIC
ASSESSMENT
OF
BONE
MECHANICAL PROPERTIES: BOTTOM-UP APPROACH
FROM THE TISSUE SCALE TO THE ORGAN SCALE
Grimal Q (1), Laugier P (1)
1. CNRS-UPMC
Introduction. One remarkable property of ultrasonic
waves (UW) is the scalability. Meaning, the spatial
resolution of measurements is scalable to the
wavelength. In particular, UW are advantageous to
investigate hierarchically organized materials such as
mineralized tissues. Samples can be investigated with
frequencies from 0.5MHz to assess overall mechanical
properties (e.g. hip strength) to 1GHz to probe intrinsic
elastic properties at the tissue level (down to micrometer
scale). This will be illustrated with recent studies.
Methods. Ten human femurs were involved in an in vitro
study. The cortical shell of the proximal femur was
assessed as a whole in through transmission at 0.5MHz
and the UW characteristics compared to the failure load.
In addition the intrinsic anisotropic elastic properties at
the millimeter scale were determined from contact
measurements at 2MHz on parallelepiped samples.
Impedance images of their surfaces were obtained from
50 MHz acoustic microscopy, from which porosity and
mineralized matrix rigidity were derived.
Results. UW propagation time was found to be predictive
of femur strength (R2=0.79). This is because UW reflects
both geometry and elasticity of the cortical shell. The
latter was found to be essentially determined by the
vascular porosity (R2=[0.73 – 0.84]). The mineralized
matrix properties were almost constant. We also
evidenced that the anisotropic elasticity can be
successfully modeled with popular continuum mechanics
homogenization schemes.
Conclusions. With different ultrasound measurements,
we have reached a clear understanding of the
relationships between the composition (porosity,
mineral) of cortical bone and elastic properties. This will
be useful for the assessment of mineralized tissues of
unknown composition. The confounding factor of sample
shape (e.g. complex shape at the femoral neck) can be
overcome by a thorough analysis UW with simulation
tools. The developed methods and models are also
applied to other mineralized tissues including growing
and healing tissues.
Keywords.
Mineralized
tissues,
ulrasound
characterization, elasticity, osteoporosis, bone
(12.O1) BIAXIAL MECHANICAL PROPERTIES OF THE
AORTIC VALVE: EFFECT OF THE HYALURONIC ACID
Borghi A (1), Carubelli I (1), Sarathchandra P (1), Chester
AH (1), Taylor P (1), Yacoub M (1)
Hyaluronic acid (HA) is an important component of the
glycosaminoglycans (GAGs) that are present in valve
leaflets. At present, little is known about the contribution
of the individual GAGs to the mechanical function of the
valve. To understand this, HA was selectively removed
from porcine aortic valves (AV) and the mechanical
properties of the valve cusps assessed.
Fresh right coronary (RC), left coronary (LC) and noncoronary (NC) AV cusps were dissected from adult pig
hearts sourced from a local abattoir. Each leaflet was
radially cut in two halves. In the RC group one half was
treated enzymatically to remove hyaluronic acid (HA)
while other half was left untreated as a control. In the LC
and NC groups both halves were treated using the control
buffer. Each specimen was cut into 5mm strips and
mounted on a BOSE electromechanical tensile testing
machine. A peak level of load equal to 1.1 N/cm was
applied. Each strip was first preconditioned to this level
with a frequency of 0.1Hz for 20 cycles. Stiffness and
percent relaxation were analysed. Alcian Blue/Sirius Red
staining was used to evaluate how efficient the enzyme
treatment was. Tinctorial staining showed that most of
the sulphated GAGs were removed from the RC cusp after
24 hours’ enzymatic incubation. Removal of HA
increaseed the percentage decay of force during
relaxation test (17 % vs 22% control vs HA respectively),
however this effect was not statistically significant
(P=0.07). The stiffness of the valves was not affected by
removal of HA (0.19 vs 0.17 N/mm control vs HA
respectively, p=0.57). The results from the LC and NC
groups showed no difference in mechanical behaviour
between the two sides of each cusp (stiffness and %
decay were 0.22 N/mm and 25% respectively) showing
there was no difference in control group between the 2
halves of the cusp.
These data suggest that HA does not contribute to the
reported effects of GAGs on the mechanical properties of
the AV. The identities of the GAGs that affect the
mechanical stiffness of the valve require further
investigations.
Keywords. Hyaluronic acid, stress relaxation
(12.O2) ULTRASONIC MONITORING AND PARAMETERS
IDENTIFICATION OF SIMULATED TISSUE CULTURE
Rus G (1), Bochud N (1), Rodríguez JM (1), Alaminos M (1),
Campos A (1)
1. Universidad de Granada
Introduction. A monitoring Petri dish is tested for realtime measurement of mechanical properties of thin
layers of tissue culture. To verify the sensitivity, a
transformation
process
is
monitored
during
approximately an hour, and validated numerically.
Methods. A layer of phantom gel of about 100 [μm]
thickness is cultured on a Petri dish. The gel suffers
consistency changes during a period in the order of
magnitude of one hour. Simultaneously, an evaporation
process is also expected. For this task, the device was
excited by high-frequency ultrasonic burst waves at a
central frequency of 20 MHz, a duration of one cycle and
an amplitude that amounts to 5 Vpp. The signal was
registered during a period of 5 [μs] and a sampling rate of
400 [MHz]. The forward problem simulation of the
experimental system is proposed using a semi-analytical
model of the ultrasonic wave interactions within the Petri
dish and gel based on the transfer matrix formalism. This
modelling includes dispersion effects associated with
relaxation processes that occur during the propagating of
the ultrasonic wave. An inverse problem (IP) is proposed
for determining the sensitivity of the mechanical
properties of the gel regarding the time evolution of the
transformation process.
Results. This propagation model, combined with the
inversion algorithm, allow to determine the time
evolution of the mechanical properties of the gel, such as
the stiffness and the attenuation coefficient, and thus to
interpret the transformation procedure.
Conclusions. The feasibility of the proposed
reconstruction procedure using genetic algorithm to
quantify consistency changes from a single measurement
is evaluated. This framework open a number of questions
to be answered in ongoing works, such as the extension
of the forward modelling to nonlinear constitutive laws.
Acknowledgements. The authors want to thank the
following institutions: SAS, Junta de Andalucía (PI-0308),
and MICINN (DPI2010-17065), for funding.
Keywords. Inverse problem, non-destructive evaluation,
ultrasonics, tissular mechanics
(12.O3)
LOW-INTENSITY
ULTRASOUND
FOR
STIMULATION OF TISSUE CULTURE
Bochud N (1), Rodríguez JM (1), Rus G (1), Alaminos M (1),
Campos A (1)
Introduction. The propagation of mechanical waves and
interaction generated with tissular microstructure has not
been addressed enough to characterize both physical
principle of diagnosis and treatment. Recent results
evidence these aspect: Ultrasound (US) technology has
been used in biotechnology for improving of cell viability
via its ability to increase mass transport, and also in the
context of cartilage and bone regeneration or tissue
engineering, where it increased cellular activity. In
accordance with them, a stimulation ultrasound wave
device is proposed. The ultrasonic wave energy,
frequency and shape is estimated to be compatible to
those used in previous references by analyzing the signal
from a receiver.
Experimental methodology. Layout of design and
methodology has been developed based on simulationoptimization of a high energy and low energy transductor,
using Finite Elements Methodology (FEM). A robust
algorithm to reconstruct mechanical parameters from
measured signals was applied. Equipment allows to
generate a variable frequency-energy-shape excitation
ultrasonic signal. The transmitted signal was generated as
a 10-cycle burst composed by a 50
− 500 [kHz] sine of
variable amplitude with a repetition rate of 10 [ms]. This
signal interacts with the culture and the interaction is
captured by the ultrasonic receiver. The received signal is
amplified, digitized with a high resolution A/D converter,
and digitally processed off-line in a computer, using
MATLAB.
Results. If a transmitted signal of frequency f = 50 [kHz] at
amplitude Dt = 10 [V] yields a registered signal of
amplitude Dr = 0.5 [mV], the stress at the tissue to be
stimulated is estimated to be of the order of 368 [Pa].
Similar magnitudes were observed in the range between f
= 20 − 500 [kHz], with a monotonically increasing trend.
Conclusions. Scientific and strategic strength lie on
ultrasounds interaction with tissue, and applied
engineering in physical devices, to face up a deep
understanding at a micromechanical scale of tissue and
physical organ level. Establishing a link between
regenerative medicine and a possible contribution during
clinical surgery.
and MICINN (DPI2010-17065), for funding.
Keywords. Inverse problem, low-intensity ultrasounds,
non-destructive testing, tissular mechanics
(12.O4) EXPERIMENTAL CHARACTERIZATION AND
CONSTITUTIVE MODELING OF THE MECHANICAL
BEHAVIOR OF THE HUMAN TRACHEA
Trabelsi O (1), Pérez del Palomar A (2), López-Villalobos JL
(3), Ginel A (3), Castellano MD (1)
1. Group of structural mechanics and material modeling
(GEMM), Aragon Institute of Engineering Research (I3A),
University of Zaragoza, Spain; 2. University of Zaragoza,
Spain; 3. Hospital Virgen del Rocío, Department of
thoracic surgery, Seville, Spain
Introduction. Cartilage and smooth muscle constitute the
main structural components of the human trachea; their
mechanical properties affect the flow in the trachea and
contribute to the biological function of the respiratory
system. The aim of this work is to find out the mechanical
passive response of the principal constituents of the
human trachea under static tensile conditions and to
propose constitutive models to describe their behavior.
Methods. Histological analyses to characterize the tissues
and mechanical tests have been made on three human
trachea specimens obtained from autopsies. Uniaxial
tensile tests on cartilaginous rings and smooth muscle
were performed. Cartilage was considered an elastic
material and its Young Modulus and Poisson Coefficient
were determined fitting the experimental curves using a
Neo-Hookean model. The smooth muscle was proved to
behave as a reinforced hyperelastic material with two
families of fibers, and its nonlinearity was investigated
using the Holzapfel strain-energy density function for two
families of fibers to fit the experimental data obtained
from longitudinal and transversal cuts. FE-simulations
were made using the experimental results to check the
influence of a tracheal implant on swallowing.
Results. For cartilage, fitting the experimental curves to
an elastic model, a Young modulus of 3.33 MPa and Nu=
0.49 were obtained. For smooth muscle, several
parameters of the Holzapfel function were found out
C10=0.877 KPa, k1=0.154 KPa, k2=34.157, k3=0.347KPa
and k4=13.889 demonstrated that the tracheal muscle
was stiffer in the longitudinal direction. The FEM results
permitted to estimate the consequences of a Dumon
stent implantation in the stress state of the trachea
during swallowing.
Conclusions. The better understanding of how these
tissues mechanically behave is essential for a correct
modeling of the human trachea, a better simulation of its
response under different loading conditions, and the
development of strategies for the design of new
endotracheal prostheses.
Keywords. Tracheal cartilage, smooth muscle, tensile
tests
(12.O5) MYOFIBROBLAST AND CARDIOMYOCYTE
INTERACTIONS STUDIED IN A MODEL SYSTEM
Abney T (1), Elson E (1), Schaefer PM (1), Pryse T (1),
Wakatsuki T (2), Genin G (1)
1. Washington University in St. Louis; 2. Medical College
of Wisconsin
Interactions between myofibroblasts and cardiomyocytes
are important to understanding the long-term
consequences of cardiac fibrosis and myocardial
infarction, but are difficult to quantify in natural tissue.
We therefore study these in an idealized model system
known as engineered heart tissues (EHTs), assembled
from embryonic cardiomyocytes and containing defined
fractions of myofibroblasts randomly distributed
throughout the tissue.
EHTs are assembled by suspending ~106 cells obtained
from 10-12 day chicken embryos in 1 ml of ~1mg/ml type
I rat tail collagen. Over several days of incubation the
primary fibroblasts convert to myofibroblasts that
compress and stiffen the collagen. Within 4-7 days the
cardiomyocytes, which begin contracting independently,
establish gap junctions and begin to beat coherently.
Then the EHT twitch force is readily measurable with an
isometric force transducer, and the spread of electrical
excitation can be measured using optical mapping
techniques. The fraction of cardiomyocytes can be varied
from ~5% to ~ 95%.
Central questions are how myofibroblasts and
cardiomocytes are coupled electrically in EHTs, and how
overgrowth of tissues by proliferative myofibroblasts
affects mechanical function. We present here progress
towards answering these questions.
Keywords. Cardiac fibrosis, myofibroblasts, engineered
heart tissue, model systems
(12.P1) STRUCTURAL AND FUNCTIONAL CHANGES IN
RABBIT CAROTID ARTERIES AFTER EXERCISE TRAINING
Benavent N (1), Machado I (1), Mauricio MD (2), Aldasoro
M (2), Vila JM (2), Noguera R (1)
1. Departments of Pathology, Medical School of Valencia,
University of Valencia, Valencia, Spain; 2. Departments of
Physiology, Medical School of Valencia, University of
Valencia, Valencia, Spain
Introduction. The response of the endothelium to
training exercise depends on a number of factors that
include the training program duration, and the size and
anatomical location of the artery.
Objectives. To evaluate whether physical training
produces histological and functional changes in rabbit
carotid artery.
Methods. Eleven rabbits were exercised for 6 weeks
following a protocol on treadmill and another twelve
rabbits were stabulated during the same period. After
exercise program, the rabbits were anaesthetized and
killed, and the carotid arteries were dissected, fixed and
included in paraffin blocks with horizontal and transversal
orientation. Hematoxylin and eosin microphotographs
were digitized and analyzed using Photoshop and Image
Proplus software. The number of the muscular layers and
the thickness of the vascular structures were measured.
To study the vascular function, arterial segments (3 mm
long) were mounted for isometric recording of tension in
organ baths containing Krebs-Henseleit solution
Results. The number of vascular smooth muscle cell
layers were similar in control and trained animals (9 to
10) but a thinning of the media layer was observed in
trained animals (77±8µ vs 65±10µ, p<0.001). Potassium
chloride (5-120 mM) induced a concentration-dependent
contraction that was lower in arteries from trained
rabbits (EC50 values: 27±2 mM for control group vs
42±4mM for training group, n=10; p<0.001). Sodium
nitroprusside, an endothelium-independent relaxant (109 to 10-6M) produced concentration-dependent
relaxation that was higher in arteries from trained rabbits
(EC50 values: 2.7x10-8M for control group vs 1.3x10-8M
for training group, n=10; p<0.05) while acetylcholine, an
endothelium-dependent relaxant, (10-9 to 3x10-6M)
produced concentration-dependent relaxation that was
lower in arteries from trained rabbits (EC50 values:
3.7x10-8M for control group vs 7.1x 10-8M for training
group, n=10; p<0.05).
Conclusion. Exercise training decreases smooth muscle
thickness, increases basal production of NO in the smooth
muscle cells and decreases NO release from the
endothelium.
Keywords. Rabbit carotid artery, exercise training,
histological changes, smooth muscle
(12.P2) SPECIFICITY OF PIEZOELECTRIC TISSUE STIFFNESS
SENSOR: MODELING
Rodríguez JM (1), Bochud N (1), Calborg GR (1)
Introduction. Reliable quantification of the stiffness
modulus of soft tissue is an open issue with relevance for
the diagnostic of pathologies that appear as drastic
changes in the consistency of the tissue, such as tumors.
The reconstruction of such parameters from non
destructive testing based on ultrasonic transmission of
pulses and model-based solution of the identification
inverse problem is proposed as a novel technique with
high potential for the direct relationship and sensitivity of
the propagation of those mechanical waves to the
mechanical stiffness of the tissue, which defines the
ultimate criterion for diagnosis.
Methods. A model-based Inverse Problem is applied to
reconstruct the values of the linear stiffness constants
that best fit the experimental measurements. Two inputs
need to be introduced: the parametrization, responsible
for which parameters of the model control the
characterization of the sought model and the
experimental measurements. The latter ones were
obtained by performing a finite-element simulation. The
experimental measurements are simulated by a finiteelement model that includes the whole implied boundary
and transducer layers effects. A gaussian noise with zero
mean and standard deviation is added to the simulated
measurements, considering several signal-to-noise ratios.
Thus, the complete wave interactions within the
specimen are described.
Results. The model-based inverse problem that governs
the theory of elasticity has demonstrated feasibility to to
reconstruct the stiffness modulus odd soft tissue.
Conclusions. This work allows (i) to validate to which
extent a one-dimensional linear-elastic model of wave
propagation is consistent to identify the full complexity of
a simulated experiment based on the multidimensional
modeling of wave propagation within soft tissue; and (ii)
to extract practical parameters for final tissue quality
assessment.
MICINN (DPI2010-17065) and AECID (A/027182/09), for
funding.
Keywords. Inverse problem, ultrasonics, non-destructive
testing, tissular mechanics
(12.P3) AN STOCHASTIC-INVERSE APPROACH TO MODEL
THE EVOLUTION OF THE MECHANICAL PROPERTIES OF A
TISSUE CULTURE
Chiachio J (1), Chiachio M (1), Rus G (1)
1. UGR
A stochastic framework is proposed to model the
evolution of the mechanical properties of a simulated
tissue culture by means of discrete-time non-stationary
Markov chains. Even under controlled laboratory
conditions a Markov-type evolution of the tissue
mechanical properties is expected, under the hypothesis
that the future of the process depends only upon its
present state, and not upon its past states. A unitary
time-transformation concept by means of monotonic
cubic Hermite splines is introduced to take into account
the nostationarity of the process. An inverse-problem
based procedure is proposed to find the optimal
stochastic model parameters together with the time
transformation parameters by minimizing a cost function
that quantifies the mismatch between experimental and
numerically predicted distribution functions. The validity
of the proposed methodology is discussed in relation to
real time experimental data. A monitoring Petri dish with
a 20 [MHz] ultrasonic transmitter and receiver is
specifically designed and stochastic mechanical data of a
culture process is taken. This approach has been tested
successfully in materials with a complex stochastic
evolution such as composites materials. As further work,
ultrasonic transmission signals are examined to be used
as raw experimental data within a Bayesian Inverse
Problem framework. In this way the accuracy of the
method is expected to improve due to the use of the
redundant data contained within the signals.
Keywords. Tissue culture, Markov chains, inverse
problem, nonstationarity, ultrasounds
13. COMMERCIALIZING CELL
THERAPIES. TRAGEDY, TUMULT AND
TRIUMPH
Chair: Brian Newsom
Keynote speakers: Eduardo Bravo, Gil Beyen
Organizer: Brian Newsom
Synopsis: Taking a cell therapy from development to
commercialization has proven to be a rocky road. Along
this road we see many that have fallen to the wayside and
there are many more that will no doubt end up with that
fate. There are some, however, that have overcome the
hurdles (the regulation, the funding, the clinical proof) to
bring their therapies to late stage trials and
commercialization. We will hear of the trials and
tribulations of those that have paved the way to success
in the area of Tissue Engineering and from those on the
brink of success.
Bringing a cell therapy to the market takes a lot more that
interesting or even useful science. It takes a lot of
dedicated people, exceptional funding (and therefore
fundraising skills), good clinical practice, attention to
detail in manufacturing, quality and logistics & and strong
case for being able to generate revenues at the end of a
10+ year development cycle. To date only 4 companies
have managed to finish this trek, and only one under the
new European ATMPs. We will hear from this company as
well as another company that may join them as the
second member of this elite group to find out how they
achieved this feat and what wisdom they can pass along
to those currently developing cellular therapies.
(13.KP1) CELLERIX: EXPERIENCES AND LESSONS IN CELL
THERAPY
Bravo E (1)
1. Cellerix
Stem cell therapy is regarded as one of the most
promising biopharmaceutical approaches currently in
development. However, the unique challenges of bringing
a “living medicine” to the market has required the
industry to address a diverse range of aspects in multiple
areas, including but not limited to:
- Advanced therapies’ regulation being put in place as
R&D advances
- Past IP makes it difficult to ensure efficacious protection
of new developments
- Challenges to be overcome in production, logistics and
later on commercialization
- Difficult financing environment (association of cell
therapy with gene therapy, lack of success cases, etc)
Cellerix is focused on the development of expanded adult
stem cells from adipose tissue (eASCs) for the treatment
of immune mediated inflammatory indications. The
Company began development in a niche indication with
an autologous product to take advantage of the clearer
regulation and faster route to market. Almost seven years
after inception, Cellerix is now starting a phase I/II trial in
the blockbuster rheumatoid arthritis indication with IV
infusion of allogeneic stem cells.
This fast advance has been possible thanks to various
strategic decisions taken by the Company that include:
- The early adoption of the clinical trial route vs. taking
advantage of unclear regulation that exempted certain
stem cell treatments from clinical trials
- The close communication with regulatory agencies on
development plans and protocols
- The investment in the development of a platform rather
that a specific product
Cellerix’ platform builds upon a well characterized stem
cell population with a common preclinical, CMC and
manufacturing package. This strategy has been
instrumental in growing the Company’s pipeline and
creating value, as it has allowed Cellerix to capitalize on
past work performed by building upon first generation
products irrespective of their clinical trial results for the
development of second generation treatments.
Keywords. Adipose Derived Stem Cells, AMTP,
Inflammatory indication
(13.KP2) CHONDROCELECT: FIRST COMMERCIAL
EXPERIENCE WITH AN ATMP
Beyen G (1)
1. Tigenix
ChondroCelect is the first Advanced Therapy Medicinal
Product (ATMP) centrally approved under the new
European ATMP regulation. ChondroCelect is an
autologous cell therapy product consisting of in vitro
expanded autologous chondrocytes, and is indicated for
the repair of damaged cartilage of the knee. This
medicinal product is currently being reviewed in several
European key countries by the national Health Agencies
responsible for pricing and reimbursement. As
ChondroCelect is the first ATMP undergoing this
evaluation, a wealth of experience in this matter is being
gained. Selected case studies in different European
countries will be presented.
Keywords. Tissue Engineering, cell therapy, ATMP,
cartilage, ChondroCelect
(13.O1) ORGANOGENESIS INC.: THE ROAD TO
COMMERCIALISATION
MacKay G (1)
1. Organogenesis Inc.
Organogenesis Inc. was an early pioneer in regenerative
medicine. The company incorporated in 1985 to develop
cell therapies originally developed at MIT. A key
milestone was achieved in 1998 when Organogenesis
received the first FDA approval for a living, allogeneic,
human cell-based product. Apligraf® has now treated
hundreds of thousands of patients and is now a standardof-care option for chronic wounds in the USA. In fact, an
Apligraf is applied to a patient Monday to Friday every
hundred seconds.
Organogenesis Inc. had difficulty transitioning from a
research based company to one with solid commercial
skills. There were little or no models to follow and several
initial approaches failed. Through this learning, a
company has emerged with the unique skill sets to take
living technology from applied research, through scaleup, to full commercialisation to medical clinics in multiple
countries.
The goal of this presentation is to highlight some of the
choices, approaches and eventual successes addressed
during the path to business success. These involve tough
R&D decisions, process approaches, automation
investments, regulatory and sales and marketing build up.
Having built up a material level of revenue, profit,
infrastructure and a skilled team, the presentation will
finish with a view of what’s next.
Keywords. Regenerative medicine, Apligraf
(13.O2) CHONDROGENIC BUT NOT OSTEOGENIC
DIFFERENTIATION OF BONE MARROW DERIVED STRO-3+
MESENCHYMAL PROGENITOR CELLS IN THE OVINE
CERVICAL SPINE
Ghosh P (1), Goldschlager T (2), Zannettino A (3),
Gronthos S (3), Itescu S (1), Jenkin G (4)
1. Mesoblast Ltd.; 2. Monash medical Centre; 3. Hansen
Institute; 4. Richie Centre, MIMR
Introduction. The objective of this animal study was to
show that adult allogeneic Stro- 3+Mesenchymal
Precursor Cells (MPC) formulated with Pentosan
Polysulfate (PPS) and embedded in biodegradable
collagen scaffolds would produce hyaline cartilage (HC)
but not bone.
Methods and Materials. Eighteen ewes were subjected
to C3/4 and C4/5 anterior cervical discectomy, followed
by the implantation of interbody cages packed with
collagen sponges with and without MPC. Group A (N = 6)
contained sponge alone; Group B (N = 6) sponge +1
million MPCs; Group C (N = 6) sponge +1 million MPCs +
10ug PPS. Radiographs of the cervical spine were taken 1,
2 and 3 months postoperatively. All animals were
sacrificed at 3 months, spines removed and scanned by
CT. For histological studies the C3/4 and C4/5 motion
segments encompassing the cages were isolated. After
decalcification and paraffin embedding, sagital sections
were cut through the cages and stained with H&E and
Alcian Blue. Using the ICRS scoring system the histological
sections were examined by a blinded observer to assess
HC and bone deposition.
Results. CT analysis demonstrated the presence of new
bone within 75% of the cages of Group A and 92% of
Group B. In equivalent regions of Group C cages
containing MPC+PPS, only 8% of the levels showed
evidence of new bone formation (p = 0.0009 versus
Group A and p = 0.0001 versus Group B). Histological
scoring confirmed that there was significantly more HC
and less bone deposited within the cages of the PPS+MPC
(Group C) compared with both Group A (p = 0.003) and
the Group B (p = 0.017).
Conclusions. This is the first in-vivo study to demonstrate
the feasibility of using formulations of MPC + PPS to
produce hyaline cartilage within a biological environment
normally conducive to the production of new bone (spinal
fusion).
Keywords. Mesenchymal stem cells, chondrogenic
differentiation, new discs, pentosan polysulfatedisc,
extracellular matrix, repair
(13.O3) SUCCESSFUL REGULATORY STRATEGIES FOR
COMMERCIALISING ADVANCED THERAPIES
Zwart I (1), Blakie R (1)
1. ERA Consulting
The European legislation concerning Advanced Therapy
Medicinal Products (ATMPs) has recently been changed in
an attempt to harmonise the regulatory requirements for
the development of ATMPs within the EU and improve
patient access to such products. Despite this attempt to
simplify the regulatory environment, only one advanced
therapy has been granted a marketing authorisation in
the EU to date. This lack of approval of cell-based
medicinal products is due to the failure of many
companies to negotiate the maze of EU legislation and
overcome the regulatory hurdles that still stand in the
way of the commercialisation of advanced therapies. In
addition, a lack of regulatory foresight for products
previously classified as transplants means that many
products that were under development prior to the
enforcement of the ATMP Regulation do not meet the
standards now required of an advanced therapy.
This session will therefore outline the current EU
regulatory framework for advanced therapy medicinal
products and assess the recent regulatory experience
with cell-based medicinal products in light of the ATMP
Regulation. The key to the success of a product is the
development of a regulatory strategy early on, alongside
interaction with the regulatory authorities in the EU
during product development. Fortunately, many common
pitfalls that have led to the delay or failure to obtain
marketing authorisation for a product can frequently be
overcome by increasing awareness of the current
regulatory climate and maximising the use of the
regulatory incentives available for advanced therapies.
Keywords. EU regulation, Marketing authorisation,
Commercialisation
(13.O4) LEGAL CHALLENGES FOR ATMP DEVELOPMENT
Stevens H (1), Verbeken G (1), Verlinden M (1), Huys I (1)
1. K.U.Leuven
Introduction. Innovative breakthroughs in medicinal
products for advanced cell or gene based therapies
(ATMPs) offer hope for unmet or unsatisfied medical
needs. Certain cell based products are already
successfully applied in therapeutic context while gene
based clinical trials offer potential for a long-term
treatment of certain monogenetic diseases. However, the
number of legislative rules and guidelines increases, as
well as the cross-border challenges in multidisciplinary
studies. The complexity, plasticity and fragility of cell and
gene based products impede the legislator to present an
exact definition for these products. It is the exact
definition that is needed in order (1) to satisfy the criteria
of quality, safety and efficacy in the Medicinal Product
legislation, as well as (2) to safeguard adequate legal
protection through the patent system.
Methods. Several patents on gene and stem cell
inventions were analyzed using in-house developed
patent landscaping methods in order to perform claim
analysis and typology. The findings were put into the light
of the recent legal evolutions in the EU and US with
respect to cell and gene patenting, substantiated by
influencing case law and doctrine.
Results. Some possible legal mechanisms are proposed as
a solution for the legal uncertainties within the domain of
cell and gene therapies.
Conclusion. The suggestions may offer new insights for
ATMP development.
Keywords. ATMP - patents - regulation
14. COMPUTATIONAL MODELING IN
TISSUE ENGINEERING
Chair: José Manuel García-Aznar
Co-chairs: Hans van Oosterwyck, Georg N. Duda
Keynote speaker: Georg N. Duda
Organizers: José Manuel García-Aznar, Hans van
Oosterwyck
Synopsis: Computational modelling is a useful tool for
research in tissue engineering that, in combination with
experiments, can increase our quantitative understanding
of understanding of underlying mechanisms, as well as for
the development of new technologies. Models allow
analyzing the influence of multiple factors that are
relevant in tissue engineering: coupling of many different
biochemical and biophysical factors at different temporal
and spatial scales (from the whole organ to the cellular
level). The development of this kind of models is also a
challenge from a computational point of view, involving
multiphysics and multiscale analysis in evolving tissues
and tissue constructs.
Specific topics in this symposium could be:
• Computational modelling of cell and tissue dynamics,
relevant for tissue engineering
• Computational models to quantify mass transport in
tissue engineering constructs
• Use of computational techniques to optimise scaffold
design
The symposium wants to demonstrate that, by combining
computational analysis with (in vitro or in vivo)
experiments, new possibilities are being created both in
terms of fundamental understanding as well as
applications.
(14.KP) MECHANO-BIOLOGY OF ENDOCHONDRAL
OSSIFICATION
–
EMPLOYING
COMPUTATIONAL
MODELING TO GAIN UNDERSTANDING OF THE
UNDERLYING MECHANO-REGULATION OF TISSUE
REGENERATION
Duda GN (1)
1. Charité - Universitätsmedizin Berlin, Julius Wolff Institut
and Center for Musculoskeletal Surgery, Germany
Using the example of bone healing, the power of
computational approaches to unravel mechano-biological
regulation principles will be demonstrated. Limitations of
such approaches and opportunities shall be presented
and discussed using comparisons of computer simulation
with histology and material characterization over a period
of regeneration. Bone healing provides an ideal model to
investigate the influence of mechanics on the biological
processes during musculoskeletal tissue regeneration.
Previously, decreased fixation stability was found to
prolong the chondral phase of healing suggesting
endochondral ossification in particular to be mechanosensitive. The aim of our analyses was to investigate
potential mechanisms regulating ossification processes
during bone healing. The finite element method was used
to estimate the local stresses and strains in the callus
initially and at 2 and 3 weeks post-osteotomy. The local
stresses and strains were then correlated with the
corresponding histological patterns of tissue formation.
Initially, strains and pressures in regions of initial bone
formation were determined to be low, regardless of the
fixation stability. At 3 weeks however, high tensile strains
were estimated on the surface of the hard callus and
coincided with regions of cartilage formation, implying a
potential role for these strains in regulating the chondral
phase of bone healing. Possible explanations for the
influence of fixation stability on the processes of
ossification during bone healing are provided.
(14.O1) AN EXPERIMENTALLY VALIDATED CYTOKINE
TRANSPORT/BINDING KINETICS MODEL FOR MODELBASED ESC BIOPROCESS DESIGN
Yeo D (1), Torii R (1), Kiparissides A (1), Xu XY (1),
Mantalaris A (1)
Embryonic stem cells (ESCs) are suitable for tissue
engineering applications due to their unlimited expansion
and differentiation potential. A bottleneck towards
implementation in clinical settings is their efficient
direction towards the intended cell type. Previously, we
established that sub-optimal nutrient/metabolite culture
conditions result in spontaneous differentiation of ESCs.
Herein, we develop a cytokine transport/binding kinetics
model and address the concentration gradients within
our 3D ESCs culture systems.
Leukemic inhibitory factor (LIF ~20kDa), is indispensable
for the self-renewal of undifferentiated murine ESC
(mESC). It binds with its receptor at a rate (KD) of 1 pM
but does not pluripotency below 0.5 pM concentration.
MESCs were encapsulated (2.5×106 cells/ml) within
alginate-gelatin hydrogels (beads) and cultured in either
static or rotating wall vessel (HARV bioreactor) fed-batch
culture systems. Following a 10 day culture, both systems
reached similar cell densities of 20-fold expansion. We
estimate the Thiele modulus (Φ) to increase from 0.11 to
0.51, reducing ligand binding activity by 13%. CFX
simulations of LIF concentration show a concurrent
reduction of bead volume able to support pluripotency
(<0.3 LIFR occupancy). Our model also demonstrates that
improved LIF transfer in HARV bioreactors lead to a 2.5x
volume reduction in comparison to static. LIF activates
JAK-STAT3 signalling, which integrates with mESC
pluripotency networks via KLF4 triggering its inhibitor
SOCS3. Relative gene expression analysis shows SOCS3,
STAT3 to be significantly lowered on day 10 in static
compared to HARV cultures corroborating our
predictions.
We present a model to elucidate growth factor
interaction within our 3D systems. Our model adapts to
fit other ESC-relevant soluble factors such as FGF4,
NODAL and BMP4 improving model fidelity. Finally, we
demonstrate that the sub-optimal delivery of growth
factors leads to reduced cardiomyogenesis owing to
premature ESC differentiation.
Acknowledgements. The authors acknowledge support
from the Department of Trade and industry (UK).
Keywords. Cytokine transport/binding-kinetics model,
3D, embryonic stem cell, bioprocessing, LIF-Jak-Stat3
signalling, thiele modulus
(14.O2) MATHEMATICAL MODELING OF CANCER
SPHEROIDS
IN
BIOENGINEERED
3D
MICROENVIRONMENTS AND TREATMENT WITH AN
ANTI-CANCER DRUG
Loessner D (1), Rizzi S (2), Byrne H (3), Flegg J (4),
McElwain S (4), Clements JA (1), Hutmacher DW (2)
1. Cancer Program, Institute of Health & Biomedical
Innovation, Faculty of Science & Technology, Queensland
University of Technology, Brisbane, Australia; 2.
Regenerative Medicine Program, Institute of Health &
Biomedical Innovation, Faculty of Built Environment &
Engineering, QUT, Brisbane, Australia; 3. Centre for
Mathematical Medicine and Biology, School of
Mathematical Sciences, University of Nottingham,
Nottingham, England; 4. Institute of Health & Biomedical
Innovation, Discipline of Mathematical Science, Faculty of
Science & Technology, QUT, Brisbane, Australia
Introduction. A critical step in the dissemination of
ovarian cancer cells is the formation of multicellular
spheroids from cells shed from the primary tumor. These
cells then spread further into the peritoneum, attaching
to the mesothelial cell layer and invading into the
underlying extracellular matrix to grow secondary tumors
which is clearly the critical step leading to poor outcome.
The objectives of this study were to establish
bioengineered
three-dimensional
(3D)
microenvironments for culturing ovarian cancer cells
biomimetically in vitro and simultaneously to develop
computational models describing the growth of
multicellular spheroids in these bioengineered matrices.
Methods. Cancer cells derived from human epithelial
ovarian carcinoma were embedded within biomimetic
hydrogels of varying stiffness and cultured for up to 4
weeks. Immunohistochemistry was used to quantify the
dependence of cell proliferation and apoptosis on matrix
stiffness, long-term culture and treatment with the anticancer drug paclitaxel.
Results. Two computational models were developed. In
the first model, each spheroid was modeled as an
incompressible porous medium, whereas in the second
model the concept of morphoelasticity was introduced to
incorporate details of the bioengineered tumor
microenvironment stresses and strains. Each model was
formulated as a free boundary problem. Functional forms
for cell proliferation and apoptosis motivated by the
experimental work were applied and predictions of both
models compared with the experimental data sets.
Conclusions. This work aimed to establish whether it is
possible to discriminate between two alternative models
of solid tumor growth on the basis of cell biological data
with respect to spheroid size, cell proliferation and cell
death. Both models simulated how the growth of cancer
spheroids was influenced by mechanical and biochemical
stimuli including matrix stiffness, culture time and anticancer treatment. Our mathematical models provide new
perspectives on future experiments and have informed
the design of new 3D studies of multicellular cancer
spheroids.
Keywords. 3D microenvironment, cancer spheroids,
incompressible porous medium model, morphoelastic
model
(14.O3) FLUID MECHANICS MODELLING OF PERFUSED
CONSTRUCTS IN BONE TISSUE ENGINEERING
Oddou C (1), David B (2), Lemaire T (1), Dantan P (3)
1. Laboratoire Modélisation et Simulation Multi Echelle
(MSME), UMR CNRS 8208, Université Paris-Est Créteil,
France; 2. Laboratoire Mécanique des Sols, Structures et
Matériaux (MSSMat), UMR CNRS 8579, École Centrale
Paris ; 3. Laboratoire Matière et Systèmes Complexes
(MSC), UMR CNRS 7057, Université Paris 7
One of the key issues in generating functional tissue in
bioreactors is to quantify and optimize the hydrodynamic
mechanical microenvironment in the vicinity of the cells
within porous scaffolds. Theoretical multiphysical and
multiscale analysis related to momentum and mass
transfer phenomena through porous media could be
proposed as an interesting tool to improve culture
technique and bioreactor design [1].
In this context, using a commercial code (Femlab® 3.1;
Comsol), a computation model was developed to solve
coupled fluid dynamics and transport equations at the
microscale of the porous implant. The major
characteristics of the complex channels (pore size around
one hundred microns, tortuosity larger than two) have
been taken into account by designing a sufficiently
simplified three-dimensional representative geometry.
Inside this element, the structure of the local flow field,
its related shear stresses distribution and nutrients
transport effects have been analyzed using dimensionless
fundamental parameters.
It was shown that, as expected for such a low Reynolds
flow mimicking experimental conditions, the velocity field
structure roughly reproduces the features of the
substrate microarchitecture. Nevertheless, an unexpected
secondary flow due to the tortuous pathway is also
observed, leading to streamlines helicity and vortical
structure of the overall flow field. Thus, at the pore scale
and for sufficiently high flow rates, the associated
convective effect in the transverse direction and the
diffusive effect become comparable. This may contribute
to a significant increase in the nutriment transport
process from the centre of the pore towards the cells at
its periphery. Moreover, a concomitant non unidirectional
and inhomogeneous repartition of viscous stresses is
obtained near the channel surface (around 1 mPa for
typical experimental conditions).
[1] Oddou et al., Hydrodynamics in Porous Media with
Applications to Tissue Engineering. In Porous Media:
Applications in Biological Systems and Biotechnology, K.
Vafai Ed., Taylor & Francis, 75-111 (2011).
Keywords. Porous Media Microfluidics Transport
Phenomena
(14.O4) MESENCHYMAL STEM CELL AGEING: AN
INDIVIDUAL CELL-BASED MODELING APPROACH
Krinner A (1), Zscharnack M (1), Stolzing A (2), Loeffler M
(1), Galle J (1)
1. University of Leipzig; 2. Fraunhofer Institute for Cell
Therapy and Immunology
Introduction. Clones of mesenchymal stem cells (MSCs)
from the same donor often differ in their in vitro
properties. This kind of heterogeneity has been suggested
to originate from an individual decline in MSC function
called ‘stem cell ageing’. For therapeutic applications of
MSCs understanding the impact of in vitro culture on this
heterogeneity is crucial.
Methods and Model. Single cell-derived clones were
generated from bone marrow-derived MSC. Their
expansion was quantified and lineage/senescence
markers were assessed. Expanded clones were
subsequently applied in differentiation assays.
Our mathematical approach builds on an individual cellbased model of MSC organization. MSC differentiation is
assumed to be a stochastic process for each individual cell
with its dynamics determined by the environment. In
parallel cell-cell interactions and proliferation were
explicitly considered to impact the spatio-temporal
organization of the populations.
‘Ageing’ is introduced by the assumption that each cell
division increases the amplitude of stem cell state
fluctuations, de-stabilising these states in the progeny.
Results. We found that single-cell derived clones of MSC
show largely distinct in vitro properties regarding
expansion and both, spontaneous and induced
differentiation. While fast expanding clones did undergo
efficient induced chondrogenic and osteogenic
differentiation, slow expanding clones lack this potential.
Interestingly, spontaneous differentiation was increased
in slow compared to fast expanding clones. Co-culture of
different clones is not associated with a growth benefit.
Our model consistently describes these experimental
findings. We demonstrate that in vitro expansion itself is
sufficient to explain the observed clonal heterogeneity
and suggest further experiments to confirm our model
predictions. First qualitative modelling results on in vivo
ageing are confirmed by CFU-F of rat MSC.
Conclusion. Our model explains the observed
heterogeneity by an ageing process and suggests that in
vitro and in vivo ageing rely on the same mechanisms.
Keywords. Mesenchymal Stem Cells, Stem Cell
Heterogeneity, Cell Plasticity, Ageing
(14.O5) PREDICTION OF OSTEOGENIC DIFFERENTIATION
STATUS OF MESENCHYMAL STEM CELLS BASED ON
IMAGE ANALYSIS COMBINED WITH BIOINFORMATICS
Matsuoka F (1), Takeuchi I (2), Sasaki H (1), Agata H (3),
Kagami H (3), Honda H (1), Kato R (1)
1. Nagoya University; 2. Nagoya Institute of Technology;
3. The Institute of Medical Science The University of Tokyo
For the industrialization of regenerative medicine, the
technology for providing both higher safety assurance
and efficient cell processing is strongly required.
However, conventional and traditional experimental
techniques were considered to be inappropriate for the
continuous quality check in regenerative medicine. Since
cells produced for therapy are limited and promised to be
pure without any testing regents. In this aspect, image
analysis is one of the few methodologies that could
estimate the final condition of implanting cells after cell
processing. There had been reports of such non-invasive
cell evaluation strategies based on cell images. However,
most of the image analysis has focused on few cell
morphologies intentionally selected by experts, and there
were no scientific reason to select such parameter.
In our research, we introduced bioinformatic analysis
strategy in the image analysis of culturing cells to select
the best combination for predicting cell quality. By
successful combination strategy with the fully-automatic
cell culture and monitoring system BioStationCT (Nikon
Instruments Inc.), we succeeded in establishing a
computer model for quantifying and predicting the
osteogenic differentiation status of human mesenchymal
stem cells. We analyzed time-lapse phase contrast images
of more than 8,000 images to extract the morphologic
features and mobility features of three individual cell lots,
and examined to predict the ALP activity and calcium
deposition rate of the future (ALP activity in two weeks
later, or calcium deposition in three weeks later). From
the image analysis combined with the regression model
analysis, we found that both biologically defined
osteogenic differentiation rates could be effectively
predicted with high accuracy by the time-lapse image
information of cells. We here propose the practical
applicability of our image analysis scheme for the noninvasive cell quality analysis for the future
industrialization of regenerative medicine.
Keywords. MSC, Osteogenesis, Prediction, Image analysis,
Bioinformatics
(14.O6) A COUPLED AGENTS-TRANSPORT MODELLING
FRAMEWORK AS A DESIGN TOOL FOR BIOREACTORS
Kaul H (1), Cui ZF (1), Ventikos Y (1)
Bioreactors serve as tools for the ex vivo development of
functional tissues and as culture model systems shedding
light on fundamental dynamic mechanisms of cell
function. Despite the technology advances, bioreactors
are still, to a great extent, utilised as black-boxes where
trial and error eventually leads to the desirable cellular
outcome. With the advent of computational techniques,
investigators have tried to recapitulate the dynamics of
tissue growth inside a bioreactor but with limited success
– mainly due to inherent assumptions and restrictions of
the modelling platforms tried.
In this study, a multi-paradigm modelling framework
combining and coupling fully an agent-based approach
with computational transport phenomena is presented,
aiming to serve as a design tool for the construction of
bioreactors. The impact of factors such as volume, cell
density, flow velocity, shear stress, mass transfer and
others, on cell behaviour can be analysed before the
actual construction of a design prototype.
To demonstrate the impact of bioreactor geometry and
initial conditions on tissue growth, and vice versa, a series
of test cases are simulated in virtuo. Three virtual
bioreactors are constructed and seeded with varying
densities of virtual cells. The virtual cells were considered
as entities governed by a set of simple rules that are
capable of displaying migration, division, proliferation,
chemotaxis and apoptosis. The rules governing the virtual
cells involve constants as well as variables; the latter
emerging from aspects of the computation simulating
mass transfer inside the bioreactors.
We conclude that bioreactor geometry and initial
conditions as well as the nature of evolving cellular
behaviour has a cumulative impact on the dynamics of
the overall tissue development and that the modelling
framework presented here can be used as a concept
selection tool during the bioreactor design process to
choose, given the desired cell phenotype, optimal
specifications.
Keywords. Bioreactors, computational modelling, tissue
engineering, agent-based modeling
(14.O7) A POPULATION BALANCE MODEL TO
INVESTIGATE THE KINETICS OF IN VITRO CELL
PROLIFERATION
Fadda S (1), Cincotti A (1)
1. Dip. Ing. Chimica - Univ. Cagliari (ITALY)
The goal of this work it to develop a novel mathematical
model helpful to investigate the kinetics of in vitro
proliferation of adherent cells. The proposed model is
based on a Population Balance (PB) approach that allows
to describe cell cycle progression through the different
phases experienced by all cell of the entire population
during their own life. Specifically, the proposed model has
been developed as a multi-staged 2-D PB, by considering
a different sub-population of cells for any single phase of
the cell cycle (G1, G0, S, and G2/M). These subpopulations are discriminated through cellular volume
and DNA content, that both increase during the mitotic
cycle. The adopted mathematical expressions of the
transition rates between two subsequent phases and the
temporal increase of cell volume and DNA content are
thoroughly analysed and discussed with respect to those
ones available in the literature. Specifically, the
corresponding uncertainties and pitfalls are pointed out,
by also taking into account the difficulties and the
limitations involved in the quantitative measurements
currently practicable for these biological systems.
To this aim, a series of numerical simulations related to
the in vitro proliferation kinetics of adherent cells is
presented. First the complex task of assigning a specific
value to all the parameters of the proposed model is
addressed, by also highlighting the difficulties arising
when performing proper comparisons with experimental
data. Then, a parametric sensitivity analysis is performed,
thus identifying the more relevant parameters from a
kinetics perspective.
Keywords. Adherent cells, proliferation, population
balance, modeling
(14.O8)
COMPUTATIONAL
SIMULATION
OF
MECHANOELECTRIC
INTERACTIONS
BETWEEN
MYOFIBROBLASTS AND CARDIOMYOCYTES IN A TISSUE
MODEL
Abney T (1), Elson E (1), Nekouzadeh A (1), Wakatsuki T
(2), Genin G (1)
1. Washington University in St. Louis; 2. Medical College
of Wisconsin
Myofibroblasts are central to the wound healing process,
serving to repair and contract wound surfaces. Under
conditions of hypertension and following myocardial
infarction cardiac fibroblasts convert from their quiescent
state to this larger and contractile phenotype that can
lead to a pathologic condition, fibrosis, involving the
formation of excess fibrous connective tissue. In both
cases, the interactions of myofibroblasts with
cardiomyocytes and their ramifications for tissue function
are uncertain. To address this, we have implemented an
integrated suite of computational models of mechanical
and electrical interactions of the two types of cells, in
parallel with an idealized extracellular matrix, and are
working to validate and refine predictions through
experiments on a model system known as engineered
heart tissues (EHTs).
The computational model is formulated at the cellular
level taking into account individual cardiomyocyte and
myofibroblasts to yield the pattern of impulse spread as
modulated by the presence of myofibroblasts acting
either as insulators or resistors. The excitatory impulse
activates the contraction of individual viscoelastic cells
that are mechanically linked to other cells and the
extracellular matrix (ECM). Three classes of models are
linked in these simulations: electrophysiologic models,
models of the contractile response of individual
cardiomyocytes as a function of their internal non-bound
calcium levels, and models linking these cellular
responses to the overall mechanics of an EHT.
The modeling objective is to predict the effects of
myofibroblasts on electrical and mechanical functioning
of EHT specimens. The typical simulation predicts twitch
forces and patterns of electrical depolarization of an EHT
with defined composition that is held isometrically and
paced electrically. We will present results that shed light
on how myofibroblasts can both improve and attenuate
the active mechanical function of EHTs.
Keywords. Cardiac fibrosis, myofibroblasts, engineered
heart tissue, model systems, electrophysiological model
(14.O9) SCAFFOLD DESIGN FOR BONE TISSUE
ENGINEERING
Dias MR (1), Fernandes PR (1), Guedes JM (1), Hollister SJ
(2)
1. IDMEC-IST, Universidade Técnica de Lisboa; 2. Scaffold
Tissue Engineering Group, University of Michigan
Introduction. In Bone Tissue Engineering, the scaffolds’
functions are to promote cell proliferation, diffusion of
oxygen, nutrients, waste products and to ensure the
required mechanical properties. Therefore, scaffolds
should have enough strength but also be highly
permeable.
The objective of this study is to develop a computational
tool for scaffold design, optimizing its performance with
respect to these requirements.
Methods. First, a computational analysis of scaffold
permeability was performed, applying homogenization
methods to Darcy Law, in order to obtain the equivalent
homogenized permeability coefficients. The analysis was
done for nine models with cylindrical pores in the three
directions (different pore sizes and porosity degrees),
designed using custom IDL programs. Then, three
examples of each model were built using Solid Free Form
techniques and tested experimentally. Finally, based on
previous study, a scaffold topology optimization model
was developed using a multicriteria formulation.
Results. The comparative permeability study shows that
the computational values were not completely identical
to the experimental ones. Nevertheless, the relations
between permeability, porosity and pore size were similar
in both cases, supporting the use of this mathematical
approach for scaffold design optimization.
With the topology optimization tool based on
homogenization methods, it was possible to obtain
structures with interconnectivity in all the directions by
maximizing permeability; structures presenting a material
distribution such that the mechanical function is
optimized by maximizing elasticity; and compromising
solutions between both criteria when using the
multicriteria formulation.
Conclusions. The computational approach assumed in
this work can be extremely useful in scaffold design for
Bone Tissue Engineering. It has demonstrated its
capability to provide solutions of microstructures able to
promote diffusion without compromising the mechanical
properties, allowing the scaffold to promote the growth
of new bone even in bearing load situations.
Acknowledgements. This work was supported by FCT,
project
PTDC/EME-PME/104498/2008
and
PhD
scholarship SFRH/BD/46575/2008.
Keywords. Bone Tissue Engineering, Homogenization,
Scaffold Design Optimization
(14.O10) A 3D MULTIPHYSIC MODEL FOR THE
PREDICTION OF ENGINEERED TISSUE GROWTH IN
PERFUSED BIOREACTORS
Laganà M (1), Mara A (1), Nava M (1), Raimondi MT (1)
1. Politecnico di Milano
An essential step toward the obtainment of functional
tissue in vitro is to control its growth process. This
depends on various space- and time-varying biophysical
variables of the cell environment, primarily mass
transport and mechanical variables, all involved in the
cell’s biological response. In the aim to obtain a
quantitative law for tissue growth in function of such
variables, we have developed an advanced growth model
of cartilaginous tissue, featuring a mini-bioreactor
system, allowing local and non-destructive assays on the
cellular constructs, interfaced to a multiphysic model of
tissue growth.
The mini-bioreactor hosts 3D cellular constructs, 400
microns in thickness, seeded with chondrocyte cells and
cultured under interstitial perfusion of the culture
medium. Time-lapse fluorescence microscopy is used to
estimate local cell and extra-cellular matrix densities,
within specific locations of the scaffold, over the time
course of culture. The biomass growth around the
scaffold fibres is modelled imposing moving boundary
conditions at the biomass surface interfaced with the
flowing medium. The boundary movement is modelled as
a function of the local oxygen concentration and fluid
shear stresses, calculated at the boundary itself.
Several aspects of the non homogeneous tissue growth
seen in vitro could be quantified with this growth model.
For example, the decrease in tissue growth during the
course of the culture, either along the flow direction, due
to progressive depletion of oxygen from the flow (See
Figure), or in areas of higher tissue volume fraction, due
to the inhibition effect of non physiological fluid-induced
shears.
Acknowledgements. This research is funded by the
grants: ‘Biosensors and Artificial Bio-systems’- Italian
Institute of Technology (IIT-Genoa); ‘5x1000-2009-HMED:
Computational Models for Heterogeneous Media’Politecnico di Milano; ‘3D Microstructuring and
Functionalization of Polymeric Materials for Scaffolds in
Regenerative Medicine’- Cariplo Foundation (Milano).
Keywords. Bioreactor, multiphysic, growth, model
(14.O11) MODELLING MECHANOSENSING IN CELLMATERIAL INTERACTION: IMPLICATIONS FOR TISSUE
ENGINEERING
García-Aznar JM (1), Sanz-Herrera JA (2), Borau C (1), Rey
R (1), Moreo P (3)
1. Universidad de Zaragoza; 2. Universidad de Sevilla; 3.
Ebers Medical Technology SL
Introduction. Cells sense the mechanical environment by
pulling on the extracellular matrix (ECM). Understanding
of how mechanical environment is able to guide cell
function (proliferation, migration and differentiation) is
fundamental for multiple tissue engineering applications.
The main purpose of this work is to explore through
computer modelling how cells interact with their
surroundings.
Methods. We construct a phenomenological model that
incorporates the main mechanical components of the cell
when it is interacting with the material:
• The active part of the cell corresponding to the
contractile acto-myosin system is simulated following a
Hill force-elongation relationship.
• The actin filaments are the main component that bear
tension and work in conjunction with the acto-myosin
system.
• The passive component of the rest of the cell is due to
the contribution of the microtubules and the cell
membrane linked to the external ECM through focal
adhesions and transmembrane integrins that are
simulated as rigid unions.
To evaluate the predictive potential of this model we
have computed different mechanical properties of the
material and with different geometrical configurations of
the substrate (planar and curved).
Results and Conclusions. After the analysis of these
simulations, predicted results are in concordance with
different experimental measurements:
• Tensional forces generated in the cell increase with the
stiffness of the material in which the cell is adhered.
• External forces modify the orientation and the forces
generated by the cell.
• Substrate curvature regulates the stress distribution in
the cell and may guide the cell polarization in the
direction of minimal curvature.
Therefore, the mechanical properties of ECM scaffolds
and its local geometry are basic parameters to mimic a
local favourable environment for tissue regeneration.
Acknowledgements. The authors gratefully acknowledge
the research support of the Instituto Aragonés de
Ciencias de la Salud through the research project
PIPAMER10/015
Keywords. Finite Element Modelling, cell mechanics,
mechanosensing, durotaxis, tensotaxis, contact guidance
(14.O12)
MODELING
AND
FABRICATION
OF
FUNCTIONALLY GRADIENT VARIATIONAL PORE IN
HOLLOWED SCAFFOLDS WITH CONTINUOUS PATH PLAN
Koc B (1), Khoda AKM (2)
1. University at Buffalo, Sabanci University; 2. University
at Buffalo
Introduction. In this paper a novel continuous toolpath
planning methodology has been proposed to control the
internal scaffold architecture with hollow feature for
tissue engineering.
Methods. Functionally gradient variational pore
architecture has been achieved with the desired pore size
and porosity by combining two consecutive slices
generated from the 3d model. Porosity architecture in
this paper is built in stacks of two consecutive layers: (i)
ruling line based zigzag pattern and (ii) concentric spiral
like pattern. Modeling of the first layer with equal area
sub-regions from ruling line represent the zigzag pattern
ensures the biological and mechanical requirement and
the consecutive circular pattern layer mainly enforces the
desired porosity of the scaffold. A continuous and
interconnected optimized tool-path has been generated
as an input for the solid free form fabrication process.
Results. Three-dimensional layers formed by the
proposed tool path plan vary the pore size and hence the
porosity based on the required biological and mechanical
properties. The proposed methodology has been
implemented in this work and illustrative example has
been provided in figure 1. Also a comparison result has
been performed between proposed design and
conventional Cartesian coordinate scaffolds which shows
the proposed method reduces design error significantly.
Moreover, sample examples are fabricated layer-by-layer
using a micro-nozzle biomaterial deposition system and
shown in figure 1.
Conclusions. The proposed methodology generates
interconnected and controlled pore size with desired
accuracy along the scaffold architecture resulting
variational porosity and a continuous deposition path
planning appropriate for SFF processes which might
address multiple desired properties in the scaffold such as
better structural integrity, improved oxygen diffusion
during cell regeneration, cell differentiation and guided
tissue regeneration.
Keywords. Continuous deposition path, scaffold
architecture, variational pore size, solid free-form
fabrication
(14.O13) COMPUTATIONAL FLUID DYNAMICS AS A
DESIGNING AND TROUBLESHOOTING TOOL FOR
MULTIPHASE BIOREACTORS: CASE STUDY IN AIRLIFT
BIOREACTORS
Paopo I (1), Xu XY (1), Mantalaris A (1)
1. Department of Chemical Engineering, Imperial College
London, South Kensington Campus, London SW7 2AZ,
United Kingdom
Computational fluid dynamics (CFD) has been adopted as
a designing or troubleshooting tool in bioprocess
especially for stem cell application in which the process
characteristics are inaccessible due to the contamination
concern. The only way to transfer from a lab-scale to a
bench-scale is using a bioreactor. Moreover, different
types of cells need different types of bioreactors to
achieve their functionality. Hence, the airlift bioreactor
has been recently used as a device to differentiate
embryonic stem cells into type II pneumocytes in the
lung. The airlift bioreactor provides a physiological
environment, which theoretically has been known to
simulate the gas-exchange interface encountered in the
lung alveoli. Airlift bioreactors require a low power input
and provide a low shear environment with good mixing.
Herein, the hydrodynamics (gas holdup, superficial liquid
velocity, and shear rate) and mass transfer (kLa, the
volumetric mass transfer coefficient) features of different
airlift designs were determined by CFD. The simulations
were based on a three-dimensional (3D) transient model,
Eulerian-Eulerian approach, and two-phase liquid/gas
model with all phases being treated as laminar flow. The
superficial gas velocity was varied from 0.001 m/s to 0.02
m/s. The O2 transfer both simulated at normoxia (21%
O2) and hypoxia (2% O2). The simulation results indicated
that the hydrodynamics were corresponded to the data
found in the literatures and the gas holdup were agreed
with our experiment validation. The CFD results also
suggested that in which range of superficial gas velocity
(ug) that we can operate without any fluctuation in term
of the hydrodynamics. In addition, the airlift bioreactor is
suitable for shear sensitive cells with high mass transfer
rate, e.g. kLa, = 180 hr-1 at ug= 0.01 m/s and normoxia
condition. The results from these simulations have been
initially utilised as a promising hypothesis to design an
airlift bioreactor for the scalable and automatable culture
in multiphase bioreactors.
Keywords. Computational fluid dynamics (CFD), airlift
bioreactor, embryonic stem cells
(14.O14) MECHANICAL PROPERTIES AND FUNCTION OF
TISSUE-ENGINEERED CARTILAGE DEPEND ON THE RATE
OF COLLAGEN AND PROTEOGLYCANS SYNTHESIS
Khoshgoftar M (1), Van Donkelaar CC (1), Wilson W (1)
1.Eindhoven University of Technology, Department of
Biomedical Engineering
Introduction. During cartilage tissue engineering (TE), the
synthesis of proteoglycans (PG's) is faster than that of
collagen. In the present study we hypothesize that this
difference in synthesis rates may be unfavorable to the
development of the implant mechanical properties. The
rationale is that fibers, which are synthesized early during
culture, resist swelling of PG’s and are pre-strained as a
consequence. Fibers synthesized at late stages do not
limit swelling and are not pre-strained. Here, we explore
numerically the effect of the relative synthesis rates of
collagen and PG’s, for post-implantation tissue strains
during loading.
Methods. A fibril-reinforced poro-viscoelastic swelling
model was used in an axisymmetric finite element model
of medial tibia cartilage (properties: see [1]), containing a
TE implant with ½ matrix stiffness, ¾ of the PG's and ¼
collagen content of the native tissue [2] (ABAQUSv6.9 (RI,
USA); Fig. 1.a). Three cases were compared in which all,
half or one-third of the fibers were synthesized early, and
the remainder was synthesized late. Fibers strains before
and after implantation under 568.75 N (gait load) were
evaluated.
Results. Pre-implantation average fiber strain increased
from 4% when all collagen fibers were synthesized early
(Fig. 1.b.top) to 5% and 7% when half (Fig. 1.b.middle)
and one-third (Fig. 1.b.bottom) of the fibers were
synthesized early. This resulted in excessive collagen
strains of 10% and 13% for the two later cases under
loading (Fig. 1.c).
Conclusion. The faster synthesis rate of proteoglycans
(PG's) compared to that of collagen during cartilage tissue
engineering is predicted to result in excessive fibers strain
post-implantation. Such excessive strain may induce
implant failure.
Acknowledgment. Funding from the Dutch Technology
Foundation STW (VIDI-07970) is acknowledged.
Keywords. Cartilage Tissue Engineering, Implant,
Synthesis Rate, Collagen
References.
[1] Wilson, W., et al, (2007), Biomechan Model
Mechanobiol, 6, pp. 43–53.
[2] Kelly, T.A.N., et al, (2006), J Biomech, 39:1489–1497.
(14.O15) A BOOLEAN NETWORK APPROACH TO
DEVELOPMENTAL ENGINEERING
Kerkhofs J (1), Roberts SJ (2), Luyten FP (2), Van
Oosterwyck H (2), Geris L (1)
1. ULG; 2. K.U.Leuven
Introduction. Developmental engineering (DE) proposes a
biomimetic approach to replace empirical TE by using
developmental pathways to increase robustness,
consistency and quality of stem cell-derived TE products.
In order to be able to direct and observe a bone TE
process in vitro the different regulators of the in vivo
ossification need to be determined. Given the complexity
and high interdependency of the signalling pathways in
endochondral ossification a Boolean approach is taken to
model the developmental process.
Methods. In this study, a large-scale literature-based
Boolean model of the regulatory network governing
endochondral ossification was developed. The model is
implemented in GINsim (Gene Interaction Network
simulation), a program geared towards Boolean
modelling.
Results. The network is able to sequentially capture the
different stable states (resting, proliferating and
hypertrophic) the chondrocytes go through as they
progress through the growth plate, which are identical to
the cell states of chondrocytes during endochondral
ossification. The prehypertrophic state was predicted to
be an unstable state at the transition between
proliferation and hypertrophy, similar to experimental
observations. In a first corroboration step, the effect of
mutations in various signalling pathways of the growth
plate network was investigated. The model was able to
successfully predict the changes in the growth plate
structure for all simulated cases.
Discussion. These first corroboration results indicate that
the proposed growth plate network provides a
comprehensive and coherent description of chondrocyte
behaviour and cell state in endochondral ossification. This
Boolean model will allow integrating multiple external
signals and determine their effect on the cell state,
providing a rationale to guide an in vitro TE process.
Acknowledgments. This work was supported by the
Special Research Fund of the University of Liège (FRS.D10/20).
Keywords. Boolean, gene network, developmental
engineering
(14.O16) HOW INTEGRINS MAY MODULATE THE
MECHANOTRANSDUCTION
BETWEEN
HYDROGEL
MATRIX AND THE CHONDROCYTES IN CARTILAGE TISSUE
ENGINEERING
Khoshgoftar M (1), van Donkelaar CC (1), Ito K (1)
1. Eindhoven University of Technology, Eindhoven, The
Netherlands
Introduction. Mechanical stimulation enhances matrix
synthesis in cartilage tissue engineering. Because this
effect is mediated by integrins [1], it is thought to depend
on cell-matrix interaction. Here, we explore how
attachment between a chondrocyte and its pericellular
matrix (PCM), embedded in agarose, may influence the
distribution of strains during axial compression.
Methods. An axisymetric biphasic multi-scale finite
element model was used. Boundary conditions of the
micro-scale model of a chondrocyte with a PCM
embedded in agarose (Figure1.a) were derived from a
simulation of 5% unconfined compression at the macroscale [2]. The two conditions simulated at the micro-scale
were: frictionless contact and tied contact between the
chondrocyte and its pericellular matrix.
Results. The results showed that cell-matrix interaction
may considerably change the micromechanical
environment in and around a chondrocyte. With cellmatrix attachment, the intracellular strain becomes more
homogeneous and peak strains are reduced, compared to
frictionless contact (Fig 1b,c: top vs. bottom). Obviously,
the actual magnitude and distribution of strains inside the
cell depends on the organization of the cytoskeleton and
other organelles, as well as on the structure of the matrix.
Regardless, the effect that cell-matrix interaction is
important for the strain field experienced by the
chondrocytes likely persists.
Conclusion. Our findings suggest that blocking integrin
binding may influence the physical signals transmitted
from the PCM to the cells. Tensile and compressive
strains are known to fluence
in
chondrocyte activity[3],
and excessive strain reduces cell metabolism[4].
Therefore, we speculate that alteration of intracellular
physical signals due to blocking integrin attachment may
explain, in part, the experimental observation that
blocking integrins modulates the effect of mechanical
loading in chondrocyte-seeded agarose cultures[1].
Acknowledgments. Funding from the Dutch Technology
Foundation STW (VIDI-07970) is acknowledged.
Keywords.
Computational
Modelling,
Mechanotransduction,
Integrin,
Cartilage
tissue
engineering
References.
[1] Kock, L.M., et al., (2009), J Biomech, 42(13):2177–
2182.
[2] Kim, E., et al., (2008), J Biomech Eng, 130(6):061009–
10.
[3] Guilak, F., et al., (1997), Basic Orthopaedic Biomech,
pp:179–207.
[4] Kurz, B., et al., (2001), J Ortho Res 19:1140–1146.
(14.O17) A COUPLED CHEMO-MECHANO-BIOLOGICAL
MODEL FOR BONE ADAPTATION
Klika V (1), Pérez MA (2), Marsik F (3), Doblaré M (2),
García-Aznar JM (2)
1. Czech Technical University in Prague; 2. University of
Zaragoza; 3. Institute of Themomechanics, Prague
Introduction. We believe that modelling of processes in
biology requires an interdisciplinary approach. This needs
to comprise biochemistry and often mechanics, or physics
in general. There were two quite different approaches to
modelling bone remodelling - Spanish group has
developed a detailed mechanical description including
the influence of damage and the Czech team has
developed a model base on biochemical knowledge of the
process control together with mechano-chemical
coupling. The main objective was to combine models for
bone remodelling into a new one that would take
advantages from both different approaches.
Methods and results. The new model posess a
constitutive relation that takes mineral content, damage,
and porosity into account. Further, complex influence of
damage is included: fatigue damage growth and repair,
how mineral content affects fatigue, and how RANKLRANK-OPG pathway is directly influenced by damage. The
mechanical stimuli is of dynamic origin which is
accordance with the current knowledge. Bone volume
fraction (Vb) is a function of mechanical stimulus
(dynamic loading) daily strain history, further it is a
function of biochemical constituents: RANKL, RANK, OPG,
Estradiol, PTH, NO, and damage affects RANKL
concentration and in turn Vb.
Conclusions. Martin proposes inhibition of bone
remodelling by loading which exactly corresponds to
behaviour of bone formation index in this model. Further,
the model estimates ash fraction, mineral volume fraction
(calcium content) and bone volume fraction in bone
which should help to understand the relationship
between bone mechanics and biology that leads to
osteoporosis.
Acknowledgements. This research has been supported by
the Czech Science Foundation project no. 106/08/0557
and by the "Programa Europa XXI de estancias de
investigación-CAI".
Keywords. Multidisciplinary modeling, bone remodeling,
bone biochemistry, damage
(14.O18) COMPUTER SIMULATION OF MANUFACTURE
AND DEGRADATION OF SCAFFOLDS
Erkizia G (1), Juan-Pardo EM (1), Aldazabal I (2), Kim GM
(1), Aldazabal J (1)
1. CEIT and TECNUN (University of Navarra), Manuel
Lardizábal 15, 20018 San Sebastián, Spain; 2. Centro de
Física de Materiales (CSIC-UPV/EHU) - MPC, P. Manuel de
Lardizábal 5, E-20018 San Sebastián, Spain
In recent years, biomedical research has been developing
new strategies to release drugs into the human body in a
controlled manner. One of these strategies is based on
the degradation of polymeric fibres that contain drugs
inside them. As the fibres degrade, the drug is released in
a controlled way into the surrounding environment. The
drug release rate changes depending on, among other
factors, the geometry of the initial microstructure of the
scaffold, the drug distribution inside the fibres and the
type of polymer used.
The aim of the present work is to parametrically generate
valid 3D models, by which the degradation can be
simulated depending on different scaffold architectures.
A specific algorithm was developed for the generation of
initial electrospun microstructures with cylindrical
geometry. In the model, the fibre trajectory was defined
as a polyline. The resulting 3D microstructures are then
discretised into small homogeneous cubic elements
(voxels). In addition, a different algorithm was developed
for the simulation of the surface degradation process. The
model is based on a Monte Carlo method, according to
which the degradation probability of a given voxel is
related to the number of solid surrounding neighbours,
thus relating fibre degradation to its surface curvature.
The inclusion of drugs inside the fibres will also allow the
model to predict the average drug release rate.
The validation of the proposed model against empirical
measurements will result in a more effective scaffold
design, taking advantage of extended in-silico
optimisation of the design parameters before starting
time-consuming empirical experiments.
Keywords. Computer simulation, scaffold design,
degradation
(14.O19) MODELLING OF NUTRIENT MASS TRANSFER
AND CELL TRANSFER AND PROLIFERATION IN
ENGINEERED VASCULAR TISSUE AND SCAFFOLD
OPTIMISATION
Elsayed Y (1), Lekakou C (1), Tomlins P (2)
1. University of Surrey; 2. National Physics Laboratory
The tissue engineering of vascular grafts is a complex
multidisciplinary science that involves the growth of
smooth muscle and endothelial cells in a supporting
scaffold in vitro in the presence of an appropriate culture
medium containing the necessary nutrients such as
oxygen and glucose, and other substances including
growth factors, antibiotics, etc. The biofabrication process
is usually developed in an ad hoc manner to determine
the optimum scaffold and processing conditions that will
ensure the adhesion of cells, their homogeneous
incorporation in the scaffold, their survival and their
further growth and functioning to produce extracellular
matrix (ECM); this can be both expensive and time
consuming, hence there is huge interest in the
development of comprehensive process models. This
work presents a mathematical model including flow of
the cell-culture medium suspension through the porous
scaffold, mass transfer of various nutrients and also of
cells with convection and diffusion terms, nutrient
consumption, cell adherence and cell motion, cell growth
and death. Various flow conditions are considered for
different types of bioreactors, static, rotating, and
perfusion bioreactors. Furthermore, a growing tissue with
a dynamically changing structure is considered starting
from the scaffold design and proceeding with local
adjacent cell layers continuously growing into the pore
spaces. The model is validated with experimental data of
smooth muscle cells growing into an electrospun and
crosslinked gelatine scaffold for which predictions are
compared to actual data for oxygen and cell
concentration gradients. Furthermore, results are
presented from computer simulations for different
scaffold parameters (porosity, pore size, fibre diameter)
and processing conditions, and suggestions are made for
the optimisation of scaffold design and biofabrication.
Keywords. Scaffold, vascular, mathematical modelling,
diffusion, flow
(14.O20) A NEW CONSTITUTIVE MODEL TO DESCRIBE
COLLAGEN REMODELING IN TISSUE ENGINEERING
APPLICATIONS
Nagel T (1), Kelly DJ (1)
Introduction. Extracellular matrix remodeling is
ubiquitous in biological tissues and their engineered
counterparts. The collagen network can remodel its
orientation and stress-free configuration related to the
transition stretch above which the uncrimped fiber begins
to bear load. Remodeling of collagen crimp has been
shown to be involved in long bone growth, contracture,
scar pathologies and collagen gel compaction among
others. It can be cell mediated or occur via cellindependent mechanisms. The objective of this study is to
develop a new continuum model to describe collagen
remodeling in terms of stress-free configurations and
angular orientations of collagenous tissues.
Methods. The deformation gradient is multiplicatively
decomposed into a remodeling tensor and an elastic part.
The resulting intermediate configuration locally describes
the stress-free state of the collagen network and allows
the definition of appropriate deformation and structure
tensors. Evolution equations are defined for the
remodeling tensor and the mechano-regulated angular
fiber reorientation.
The model is applied to fibrin cruciform and collagen gel
compaction, cartilage tissue engineering and remodeling
of periosteum held at fixed lengths.
Results. The model successfully predicted the compaction
of collagen gels and the associated anisotropy that occurs
within such constructs along with their developing shape.
The simulations of periosteum adaptation captured the
temporal changes in force-deformation behavior.
Dynamic compression was predicted to influence the
developing mechanical properties of tissue engineered
cartilaginous constructs by affecting the collagen
organization.
Conclusion. Understanding how mechanical signals
regulate shape, organization and mechanical properties
of engineered soft collagenous tissues can be greatly
facilitated by computational models. The presented
framework allows in silico investigation of a large variety
of collagen remodeling related phenomena in both tissue
engineering and regenerative medicine. Critically the
model
successfully
captures
mechano-regulated
structural aspects such as orientation and natural
configuration. Extension is directed towards the
regulation of ECM constituent concentrations.
Acknowledgments. IRCSET, SFI
Keywords. Collagen, remodeling, hydrogel, fibrin
(14.P1) ENHANCING EMBRYONIC STEM CELL EXPANSION
BY THE COMBINATION OF PERFUSION FEEDING AND
BIOPROCESS MODEL DESIGN
Yeo D (1), Kiparissides A (1), Pistikopoulos E (1),
Mantalaris A (1)
1. Department of Chemical Engineering, Imperial College
London, SW7 2AZ United Kingdom
Embryonic stem cells (ESC) are suitable candidates for
regenerative medicine due to their high proliferative and
differentiation potential. A bottleneck to their usage is
the formation of differentiation by-products such as
teratomas which necessitates the implementation of
efficiently directed culture protocols. Cell culture
variables have been shown to strongly affect ESC
pluripotency levels. We investigated the effects of
metabolic stress (levels of nutrients and metabolites suboptimal to ESC metabolism) to ascertain their impact on
ESC expansion.
Murine ESCs were expanded in batch cultures and a
multi-scale bioprocess model was developed to analyze
their cellular kinetics, basic metabolism and gene
expression. We observed growth kinetics typical of batch
cultures and showed that ESCs differentiated even in the
presence of sufficient growth factors. Our mathematical
model predicts the emergence of a differentiated
population due to persistent exposure to inhibitory levels
of metabolites during the latter stages of the culture.
Thereafter, perfusion feeding operation eliminated this
metabolic stress enabling the maintenance of a 16-fold
total expansion from seeding density. The different
metabolic characteristics for perfusion feeding
necessitates changing 6 in 29 model parameters. We
observe the expression of pluripotency - related genes in
conjunction with a decrease in differentiation levels was
unaccounted for by growth factor availability.
Furthermore, our mathematical model also predicted the
preferential propagation of ESCs in a naive state at the
expense of differentiated cells in the metabolically
favourable conditions.
We contend that the metabolic well-being of ESC cultures
supersedes the effects of growth factors in determining
pluripotency levels based on the contrasting behaviour
we observed in perfusion and batch feeding cultures.
Furthermore, the use of model-based design of
bioprocesses provides insights into ESC pluripotency and
metabolism, thereby facilitating the development of
optimized culture protocols.
Keywords. embryonic stem cells, mathematical
modelling, stem cell bioprocess, perfusion feeding,
pluripotency, tissue engineering
(14.P2)
RAPID
MANUFACTURING
OF
THREEDIMENSIONAL RESORBABLE SCAFFOLDS FOR THE TISSUE
ENGINEERING OF HUMAN HEART VALVES
Lueders C (1), Brossmann C (2), Jastram B (2), Schwandt H
(2), Hetzer R (1)
1. German Heart Institute Berlin; 2. TU Berlin
Introduction. In the past 3 years rapid prototyping has
been further developed into rapid manufacturing, a
process allowing not only the establishment of real 3-D
models as prototypes but also the fabrication of 3-D
objects. Rapid manufacturing provides the novel
opportunity to generate real 3-D objects with product
quality directly from computer-based manufacturing
processes and to establish absolutely novel applications.
Using appropriate rapid manufacturing processes custommade human heart valve scaffolds should be fabricated
and additionally seeded with vascular cells from human
umbilical cords.
Methods. Protocols were established to collect 3-D data
from healthy heart valves using computed tomography or
magnetic resonance imaging. After model generation a
standardized segmentation and reconstruction logarithm
was generated and biocompatible casting molds for
selective laser sintering were constructed with the latest
visualization technologies. Subsequently, the optimal
parameters of biocompatible materials and the
associated production technique were developed and
established. Scaffolds produced were analyzed for
capability of cell seeding and quality controls were
performed using a contactless stripe-light scanner.
Results. Using custom-designed segmentation and
reconstruction software 3-D data of a heart valve from a
healthy male were processed to generate a 3-D model. A
3-D printer for selective laser sintering was used to
fabricate the heart valve scaffold consisting of a flexible
synthetic material. Recently, different resorbable
polymeric granules and powders based on polyglycolic
acid (PGA) and polylactide acid (PLA) have been analyzed
for the fabrication of heart valve scaffolds using the rapid
manufacturing process.
Conclusion. In general, direct 3-D printing of human heart
valve scaffolds using specific software and laser sintering
systems is feasible. With regard to the future clinical
application of tissue engineered human heart valves,
rapid manufacturing provides the crucial step from a
model to a suitable product.
Keywords. Rapid manufacturing, heart valves, resorbable
scaffolds, tissue engineering
(14.P3) MULTI-SCALE FINITE ELEMENT STUDY BASED ON
IN VIVO DATA TO EVALUATE BIOMECHANICAL
STIMULUS IN BONE SCAFFOLD
Roshan-Ghias A (1), Terrier A (1), Pioletti D (1)
1. Laboratory of Biomechanical Orthopedics-EPFL
A micro-FE analysis was developed allowing us to
evaluate the mechanical stimulation in a bone scaffold
inserted in a rat condyle when external load is applied to
the leg of the rat. The developed model corresponds to
an in vivo study.
Both distal femoral condyles of Wistar rats were operated
and a PLA based scaffold was implanted inside the hole.
Three days after the surgery, the loading (10 N at 4 Hz for
5 minutes) of the right knees started. The bone formation
was quantified using a SkyScan 1076 in vivo micro-CT
scanner. A rat femur geometry was imported in ABAQUS
for numerical analysis. The strain of the scaffold was then
calculated and was used as boundary conditions for the
micro-FE model of the scaffold. A scaffold similar in size
and architecture to those implanted was scanned. An inhouse Matlab script was used to convert the thresholded
images into 8-noded cubic finite element mesh for
ABAQUS. It was assumed that all pores are filled by
granulation tissue. The strain values were computed for
each element of granular tissue and the octahedral shear
strain was calculated as the mechanical stimulus.
The 10 N load applied on the rat femoral condyle resulted
in average largest principal strain of 620 με in the
scaffold. The average maximum principal strain in scaffold
tissue was 0.14±0.11% (Fig. 1a). The average octahedral
strain in granulation tissue was 1.2±0.8% (Fig. 1b).
In summary, we used a multi-scale finite element
modeling to estimate the biomechanical stimulus in our
rat distal femur model which resulted in enhanced bone
formation inside scaffold. We found out that strain as
high as 1.2% in the granulation tissue is osteogenic. This is
of practical use when designing scaffolds for bone tissue
engineering in load-bearing situations.
Keywords. In vivo test, µCT, µFEM, bone
(14.P4) AN INTEGRATIVE MODEL BASED APPROACH TO
OPTIMIZE CALCIUM PHOSPHATE SCAFFOLD-STEM CELL
COMBINATIONS
Carlier A (1), Chai YC (1), Moesen M (1), Schrooten J (1),
Van Oosterwyck H (1), Geris L (1)
1. K.U.Leuven
Introduction. Experimental evidence indicates a key role
for calcium ions (Ca2+) in mesenchymal stem cell (MSC)driven bone formation in calcium phosphate (CaP)
scaffolds. This study aims to develop a computational
model of MSC-driven bone formation in CaP scaffolds
with an emphasis on the role of Ca2+.
Methods. The mathematical model describes the
temporal evolution of the densities of MSCs, osteoblasts,
osteoid, mineralized bone and the concentrations of Ca2+
and a generic, osteogenic growth factor by means of
differential equations (1D). The model parameters were
derived from in-house in vitro experimental data and
literature. The set of non-linear delay differential
equations was solved using Matlab (The MathWorks, Inc.)
for several biologically relevant initial conditions and
compared to published in vivo data.
Results. The mathematical model predicted 31% bone
formation at 90 days post implantation, which agreed
well with experimental data. The model also predicted
the absence of bone formation in the case of insufficient
cell seeding or scaffold decalcification. Moreover, the
model shows that a low initial MSC density requires a low
calcium release rate, while a high initial MSC density
requires a high calcium release rate in order to maximize
the amount of bone formation. Furthermore, this
optimization window is narrow for low initial MSC
concentrations.
Conclusion. A mathematical model of the effect of Ca2+
on cellular activities and MSC-driven bone formation was
developed and verified by means of in vivo data. The
results obtained in this study suggest that this model can
be used as a tool to design and optimize CaP scaffolds in
tissue engineering applications. In the future the model
could be refined with additional cell and tissue types,
allowing for an in silico triage of cell-customized
biomaterials.
Acknowledgements. Aurélie Carlier is a PhD fellow of the
Research Foundation Flanders (FWO-Vlaanderen). This
work is part of Prometheus.
Keywords. Bone tissue engineering , calcium, calcium
phosphate, modeling
(14.P5) A COMPUTATIONAL SOLID AND FLUID
MECHANICAL ANALYSIS OF CAD- VERSUS MICRO-CTBASED MODELS OF REGULAR Ti6Al4V SCAFFOLDS FOR
BONE TISSUE ENGINEERING
Truscello S (1), Kerckhofs G (2), Moesen M (2), Torcasio A
(1), Van Bael S (1), Van Lenthe GH (1, 3), Schrooten J (2),
Van Oosterwyck H (1)
1. Division of Biomechanics and Engineering Design,
K.U.Leuven; 2. Department of Metallurgy and Materials
Engineering, K.U.Leuven; 3. Institute for Biomechanics,
ETH Zurich
Introduction. Osteogenic cell behaviour can be influenced
by both the local strain distribution (under mechanical
loading) and the fluid flow inside bone tissue engineering
(TE) scaffolds. In addition, fluid flow enhances the
transport of nutrients and soluble factors in general.
Thus, characterization of these properties is key in TE
scaffold evaluation. As the solid and fluid mechanical
properties of produced TE scaffolds may differ from the
design values due to additive manufacturing production
constraints, this study evaluated the importance of this
potential difference using finite element analysis (FEA)
and computational fluid dynamics (CFD) analysis on (i) a
computer-aided design (CAD) unit cell model and (ii) a 3D
micro-CT-based model. Simulation results were also
compared to experimental measurements.
Methods. A selective laser melted Ti6Al4V scaffold was
used as a test case. Micro-CT images were generated
(12.5 µm voxel size) without mechanical loading for FEA
and CFD analysis and (ii) at different discrete loading
steps, using in-situ compression, for experimental local
strain mapping. The apparent stiffness and compressive
strength were determined experimentally using
compression tests and a dedicated set-up was used to
evaluate experimentally the permeability. The CAD-based
and micro-CT-based computed apparent stiffness, local
strain distribution, permeability and wall shear stress
(WSS) distribution were compared.
Results. A good agreement was found between simulated
(CAD-based, micro-CT-based) and measured permeability
and apparent stiffness values. The difference between
the average WSS as predicted by the micro-CT and CADbased CFD model was 13% (Fig. 1). Surface
inhomogeneities inherent to the production process were
captured in the micro-CT-based but not in the CAD-based
model. However, they did not influence the local
properties significantly.
Conclusions. For the determination of both the global
and local solid and fluid mechanical properties, within the
investigated dimensional scale window, CAD-based
modelling performs as well as micro-CT-based modelling
and has lower computational requirements.
This work is part of the Prometheus, the Division of
Skeletal Tissue Engineering of K.U.Leuven.
Keywords. Bone tissue engineering, computational fluid
dynamics, finite element analysis, regular scaffolds
(14.P6) THE INFLUENCE OF THE ELECTRON BEAM ON
NANOLAYERS ANALYSIS
Miculescu F (1), Jepu I (2), Posornicu C (2), Lungu CP (2),
Miculescu M (1), Stancu M (3), Bojin D (1)
1. Politehnica University of Bucharest; 2. National
Institute for Laser, Plasma and Radiation Physics,
Bucharest-Magurele; 3. Petroleum-Gas University of
Ploiesti
Introduction. SEM is one of the most popular tools used
for the thin films’ characterization. In the field of SEM, the
use of simulation programs for the electron beam-sample
interactions enables the visualization of the interaction
volumes between accelerated electron beams and
samples.
Methods. The analyzing programs lies the possibility of
the planning and interpretation of the imaging (SEM) and
microanalytical Results. In this study we used CASINO®
software. for investigation of the effect of electron beam
energy on the penetration depth using SEM / EDS analysis
of Cu-Ni-Cu-Fe-Ta multilayer structures with different
thicknesses deposited by Thermionic Vacuum Arc onto Si
wafers.
Results. The influence of electron beam accelerating
voltage, ranging from 5 to 30 kV, on multilayer structures
with total thickness of 38 nm and 1220 nm prepared by
TVA, has been studied. When nanolayers are analyzed, it
is recommended the usage of different acceleration
voltages, in order to excite at least the K lines of the low
elements and L lines of the heavy elements. At low
energy of the incidence electrons, the absorption effect is
less important, because the interaction volume is closer
to the surface.
Conclusions. At a higher beam energy, the electron beam
can penetrate deeper and an intense signal of Si substrate
can be detected. The metal layer thickness is in an almost
linear relationship with the energy required for electron
beam penetration. Based on the experimental results and
mathematical models applied in Cu-Ni-Cu-Fe-Ta
multilayers study, relations between the detected signal
intensity function of the incidence electron beam
acceleration voltage were established. The simulation
results are in good agreement with experimental results.
Acknowledgements. Authors recognise financial support
from
the
European
Social
Fund
through
POSDRU/89/1.5/S/54785 project: “Postdoctoral Program
for Advanced Research in the field of nanomaterials”.
Keywords. nanolayers, SEM/EDS, volume of interaction,
computer simulation
(14.P7) SIMULATIONS OF CELL SEEDING USING
PARTICLES CODE AND RAPID PROTOTYPING SCAFFOLD
Olivares AL (1), Lacroix D (1)
1. Institute for Bioengineering of Catalonia (IBEC),
Barcelona, Spain
The control of cell seeding is critical for the development
of functional tissue engineering products. This study
presents a novel methodology to predict the cell
distribution after seeding. The optimum experimental
time, concentration of cells, scaffold microstructure and
hydrodynamic environment are the principal parameters
that can be controlled in this model. In addition, the
model is capable to determine the specific position of
cells on the scaffold wall after cell seeding. The simulation
was validated against in vitro experimental under
perfusion conditions. Based on rapid prototyping scaffold
fabricated by stereolithography, a scaffold with different
pore size in the radial direction of the cylindrical samples
was modelled. Human articular chondrocytes (HAC) were
suspended in culture medium, and seeded under
oscillating perfusion fluid flow.
An Eulerian-Lagrangian model was used to simulate the
multiphase phenomenon (cells and culture medium) and
cell adhesion conditions were applied. The cell adhered
after seven cycles in the simulation for a central section
was compared with the threshold z-stack confocal images
for cell-seeded in the in vitro experiment (Figure 1). A
similar distribution was obtained showing the clustering
of cells in the central part of scaffold and poor adhesion
on the periphery. This relation is attributed to the
distribution of pores in radial direction. Although the
model shows some very good similarity with the
experimental results, the model remains very simple. In
particular effort must be put into a more precise
simulation of cell attachment and separation as a function
of wall shear stress and of biological affinity with the
biomaterial surface. Nonetheless in this study we find
that the pattern of transportation of cells is a determining
factor in the final cell distribution and that it is strongly
dependent on the distribution of available surface area.
Figure 1: a) Distributions in particles attached through the
models in 1mm thickness and b) threshold z-stack
confocal images (500 µm thickness) of cell-seeded on
experiment c) The shear stress distribution is shown in a
cross section of the scaffold.
15. ENGINEERED HYDROGELS (AND
STEM CELLS) FOR TISSUE
REGENERATION
Chair: Manuela Gomes
Co-chairs: Rui L. Reis, Ali Khademhosseini
Keynote speaker: Ali Khademhosseini
Organizer: Manuela Gomes
Synopsis: The continuous technological developments in
the areas of micro and nanofabrication has allowed for a
finer control over the architecture of scaffolds for Tissue
Engineering applications. However, being micro and
nanotechnologies such young scientific areas in the field
of TE, it is expectable that their development is still in an
early stage. Its state of development is still mostly limited
to the top-down approach in which small products are
created with the help of large devices. Nonetheless,
several researchers have been studying the combination
of top-down and bottom up approaches for the
development of microgel units (top-down), which are
then assembled (bottom-up) to generate a tissue
construct. These microengineered hydrogels constitute a
very interesting approach for obtaining 3D tissue like
structures, enabling the possibility to control materials
properties such as adhesiveness, stiffness, cell signaling
potential, size and shape. Additionally these
microengineered hydrogels may be designed to
incorporate biomolecules, enabling the additional
function as drug or gene carrier systems. Several
polymers have been proposed for building such
structures, including natural origin polymers.
An additional important issue in any tissue engineering
approach is the need for an appropriate stem cell source,
but independently of the selected source, these will
always require an appropriate 3D environment, in a great
extent dictated by the 3D scaffold, to proliferate and
differentiate in the desired phenotype. The physical
microenvironment can be tailored through the fabrication
of microengineered structures that aim to mimic the
micro/nanoscale environment of tissues. These highly
organized and cooperative micro/nanoscale building
blocks can assemble in a controlled way to ultimately
build functional tissue substitutes. This syposium is
expected to provide an overview of recent work on the
development microengineered hydrogels with the ability
to direct stem cells behavior through nano/micro design
features combined with the controlled release of
biological molecules and hence obtaining highly
functional tissue engineered substitutes.
(15.KP) MICROENGINEERED HYDROGELS FOR STEM CELL
BIOENGINEERING AND TISSUE REGENERATION
Khademhosseini A (1,2,3)
1. Center for Biomedical Engineering, Department of
Medicine, Brigham and Women’s Hospital, Harvard
Medical School, Cambridge, MA; 2. Harvard-MIT Division
of Health Sciences and Technology, MIT, Cambridge, MA;
3. Wyss Institute for Biologically Inspired Engineering,
Harvard University, Boston, MA
Micro- and nanoscale technologies are emerging as
powerful tools for controlling the interaction between
cells and their surroundings for biological studies, tissue
engineering, and cell-based screening. In addition,
hydrogel biomaterials have been increasingly used in
various tissue engineering applications since they provide
cells with a hydrated 3D microenvironment that mimics
the native extracellular matrix. In our lab we have
developed various approaches to merge microscale
techniques with hydrogel biomaterials for directing stem
cell differentiation and generating complex 3D tissues. In
this talk, I will outline our work in controlling the cellmicroenvironment interactions by using patterned
hydrogels to direct the differentiation of stem cells. In
addition, I will describe the fabrication and the use of
microscale hydrogels for tissue engineering by using a
‘bottom-up’ and a ‘top-down’ approach. Top-down
approaches for fabricating complex engineered tissues
involve the use of miniaturization techniques to control
cell-cell interactions or to recreate biomimetic
microvascular networks within mesoscale hydrogels. Our
group has also pioneered bottom-up approaches to
generate tissues by the assembly of shape-controlled cellladen microgels (i.e. tissue building blocks), that resemble
functional tissue units. In this approach, microgels were
fabricated and seeded with different cell types and
induced to self assemble to generate 3D tissue structures
with controlled microarchitecture and cell-cell
interactions.
(15.O1) FABRICATION OF HYDROGEL FIBER BUNDLES
FROM ASSEMBLY OF POLYELECTROLYTES
Coutinho DF (1), Sant S (2), Shakiba M (3), Gomes ME (1),
Neves NM (1), Reis RL (1), Khademhosseini A (2)
1. 3B's Research Group; 2. Harvard-MIT; 3. University of
Toronto
In many natural tissues, fibrils align in parallel and closely
pack into three-dimensional (3D) hierarchical bundles of
fibers. These fibers provide tensile strength to the various
tissues such as heart, brain, bone or skin. Given their
importance in tissue function, the engineering of these
hierarchical features into materials is therefore of
biomedical relevance. Numerous strategies for the
development of a synthetic fiber bundle have been
proposed, such as electrospinning or extrusion of
polymers into aqueous solutions. However, most of the
existing techniques fail to replicate simultaneously, the
hierarchical architecture of these tissues and the
microenvironmental physical and chemical cues. Thus,
the aim of this work was to engineer hydrogel fibers that
both mimic the natural architecture of the fiber bundles
and enable the encapsulation of cells. Fiber bundles were
fabricated by polyionic complexation between cationic
chitosan (CHT) and anionic methacrylated gellan gum
(MeGG) that occurred in a polydimethyl siloxane (PDMS)
channel. The fibers were then collected and stabilized by
photocrosslinking the MeGG. The resulting architecture of
the fiber bundles was studied with atomic force and
scanning electron microscopy. Each bundle was
approximately 100 µm in diameter and contained small
fibers that were 1-5 µm in diameter. Confocal microscopy
of the hydrogel fiber bundles engineered with FITClabeled CHT showed homogenous distribution of CHT
throughout the fiber bundles. Their stability was
maintained in phosphate buffered saline over a period of
one week. A closer system to biological matrices was
achieved by covalently incorporating the adhesive motif
RGD in the MeGG backbone. Furthermore, encapsulated
cardiac fibroblasts adhered to and spread along the fibril
direction. This system combines polyelectrolyte
complexation and fluidics technology to engineer
hydrogel fibers that closely mimic the natural architecture
of fiber bundles and may be beneficial for various tissue
engineering and regenerative medicine applications.
Keywords. Hydrogel, microfibers, cell encapsulation,
tissue engineering
(15.O2) ENGINEERED STARPEG-HEPARIN HYDROGELS
ARE EFFECTIVE MULTI FACTOR DELIVERY MATRICES TO
PROMOTE ANGIOGENESIS
Freudenberg U (1), Zieris A (1), Chwalek K (1), Prokoph S
(1), Levental KR (1), Welzel PB (1), Werner C (1)
1. Leibniz Institute of Polymer Research, Dresden,
Germany Technische Universität Dresden, Center for
Regenerative Therapies Dresden, Dresden, Germany
Introduction. Effective vascularization is a prerequisite for
the success of various different tissue engineering
concepts. While short time delivery of various growth
factors has been shown to boost angiogenic response the
therapeutic more relevant long time delivery of signal
molecules from biomaterials is still a major challenge. To
address this issue we present here a novel biomimetic
material in which the high affinity of the polysaccharide
heparin was utilized to design a highly efficient release
matrix for several cytokines.
Materials and Methods. Modular StarPEG heparin gels
were synthesized, characterized and subsequently the
uptake and release of signal molecules (VEGF, FGF-2)
were studied applying Enzyme-linked immunosorbent
and radiolabeling techniques. Pro angiogeneic response
was studied in vitro using human umbilical vein
endothelial cells (HUVECs) and in vivo using a chicken
embryo chorioallantoic membrane (CAM) assay.
Results. As the utilized gels contain high quantities of
heparin, loading and subsequent release of both
cytokines occurred independently from each other and
could be tuned to customized release profiles. The
combined delivery of FGF-2 and VEGF through these
matrices resulted in pro-angiogenic effects in vitro (study
of cell adhesion, survival/proliferation, morphology and
migration) and in vivo (quantification of CAM
vascularization) being clearly superior over those of the
administration of single factors.
Conclusions. This study demonstrated that modular
starPEG-heparin hydrogels could be successfully utilized
for the combined immobilization of large quantities of
FGF-2 and VEGF and permitted an independent, tunable
delivery of both growth factors. In in vitro and in vivo
experiments combined FGF-2 and VEGF delivery exerted
superior effects on cell behavior and the angiogenic
response when compared with the provision of single
cytokines. As such, the starPEG-heparin hydrogels
performed outstandingly as an effective cytokine delivery
matrix, allowing for the application in multi-factor
settings essential for effective regenerative processes.
Acknowledgments. The work was supported by grants
from the European Commission Seventh Framework
Programme in the project Angioscaff (NMP-LA-2008214402), the European KidStem network, and DFG grants
(WE 2539/7-1 and EXC CRTD).
Keywords. Biohybrid hydrogel, heparin, growth factor
release, HUVECS, VEGF, FGF-2
(15.O3) IMMOBILIZATION OF BIOMOLECULES ON
HYDROGEL SURFACES WITH DIFFERENT STIFFNESSES
FOR THE MODULATION OF (ADULT) STEM-CELL FATE
Zouani OF (1), Kalisky J (1), Ibarboure E (2), Labrugère C
(3), Mehdi A (4), Durrieu MC (1)
1. INSERM; 2. LCPO ; 3. ICMCB; 4. CMOS
Introduction. Microenvironment elasticity and extreme
surface conditions appear important in stem cell lineage
specification (1). Here we propose a protocol for the
immobilization of different biomolecules that contain N
termini groups corresponding to a wide range of surface
matrix elasticity. We describe the synthesis of a copolymer of acrylamid and acrylic acid with different
elasticities ranging from 0.5 to 70 kPa and then the
covalent attachment of biomolecules directly without
spacers. This stiffness range is considered important for
stem cell fate (1). Cell behavior can be achieved in the
presence
of
adhesion
or
induction-promoting
biomolecules.The approach should be a suitable method
for the study of stem cell differentiation in different
lineages with multifactor variation. Thus, generation of
materials to direct stem cell fate holds potential for tissue
engineering.
Materials and Methods. Poly(acrylamyde-co-acrylic
acid)/polyacrylamide hydrogel was prepared. Elastic
modulus was measured with Dynamic Mechanical
Analysis (DMA). Peptide immobilization was performed
following the procedure described previously (2).
Functionalized hydrogels were characterized with X-ray
photoelectron spectroscopy and high resolution microimager. For this study, human mesenchymal stem cells
(from LONZA) were used.
Results. Three parameters were studied on the different
microenvironments of functionalized surfaces. Fist, the
change in cell shape was observed. Then we evaluated
the osteogenesis gene markers. Finally, cells were stained
with lineage-specific labeled antibodies: neurogenesis
with anti- β3 tubulin and osteogenesis with anti-runx2. An
example of direct effect on the fate of adult stem cells is
the sensitivity of these cells to their neuronal
differentiation in contact with a soft matrix (0.1-1 kPa).
Whether this takes place or not depends on the
biomolecule grafted onto the surface of this matrix. We
grafted RGD peptide or BMP-2 mimetic peptide (3) on
these soft matrices (3.21kPa) and we observed that stem
cells have become different after 96h of culture.
Conclusion
The results of this study suggest that “precommitting”
stem cells to a specific lineage via in vitro matrix
conditions is multi-factorial.
Acknowledgments. This work was supported in part by
the “Région Aquitaine” as well as the “Agence Nationale
pour la Recherche” (ANR) and Advanced Materials in
Aquitaine (GIS).
References.
1. Engler AJ et al. Cell. 2006;126(4):677-89.
2. Chollet C et al. Biomaterials. 2009;30(5):711-20.
3. Zouani OF et al. Biomaterials. 2010;31(32):8245-53.
Keywords. hydrogel, surface modification, stem cells,
differentiation
(15.O4) HYDROGEL-BASED MICROFLUIDICS FOR TISSUE
ENGINEERING
Tocchio A (1), Martello F (2), Tamplenizza M (3), Gassa F
(2)
1. European School of Molecular Medicine (SEMM), IFOMIEO Campus, Via Adamello 16, 20139 Milano (Italy); 2.
Fondazione Filarete, Viale Ortles 22/4, 20139 Milano
(Italy); 3. C.I.Ma.I.Na., Dipartimento di Fisica, Università di
Milano, via Celoria 16 – 20133, Milano (Italy); 4.
C.I.Ma.I.Na., Dipartimento di Scienze Molecolari Applicate
ai Biosistemi, Università di Milano, Via Trentacoste 2,
20134 Milano (Italy)
Introduction. One of the major limitations in tissue
engineering is the lack of proper vascularization.
Nowadays skin and cartilage grafts are successfully used
in-vivo mainly thanks to their low requirement for
nutrients and oxygen that can be met by the host's
vascularization. However this approach fails when applied
to complex and massive tissues. The formation of new
blood vessels is indeed a slow phenomenon and the
deficiency of oxygen and nutrients supply rapidly cause
widespread cell death in the graft's core. With the aim to
overcome this hindrance we developed an innovative
technique based on sacrificial elements. In this approach
fluidics channels are deeply embedded within the
hydrogel scaffold in order to favor biomimetic synthetic
vasculature generation.
Methods. The sacrificial structure of polysaccharides is
fabricated by injection molding. Murine fibroblasts (NIH3T3) are encapsulated into a liquid hydrogel matrix
(PEGDA-RGDS) and cast around the sacrificial structure,
suspended in a mold. After the UV hydrogel crosslinking,
the sacrificial template is dissolved in PBS forming
interconnected channels inside the cell-laden hydrogel.
The construct is incubated and perfused with culture
medium (DMEM) for three days. Live/Dead assay is
performed for cell viability analysis. HUVEC cells are then
cultured in microchannels and CD31 fluorescence staining
is performed.
Results. In the core sections of cell-laden hydrogel,
cultured in static condition, the cell viability decreased
with time, achieving cell death after 72 hours of in vitro
culture. In perfused microfluidic hydrogel, cell viability is
significantly higher than in static control (Fig. 1). CD31
staining confirmed rudimental endothelial tubule
formation.
Conclusion. This technique allows a high diffusion rate of
nutrients and oxygen throughout the hydrogel scaffold.
Further developments are required to generate a
“biomimetic synthetic vasculature”, mostly combining
prefabricated vessels with controlled blood vessel–
recruiting growth factors to induce growth of functional
vascular network.
(15.O5) PRODUCTION OF ENGINEERIZED ALGINATE
BASED MICROCAPSULES FOR CELL IMMUNOISOLATION
CONTAINING EXTRACELLULAR MATRIX COMPONENTS
Mazzitelli S (1), Johnson S (2), Badylak SF (2), Nastruzzi C
(3)
1. Dep. Biochemestry and Molecular Biology, University of
Ferrara, Ferrara, Italy; 2. McGowan Institute for
Regenerative Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania; 3. Dep. of Pharmaceutical
Sciences, University of Ferrara, Ferrara, Italy
This paper reports the production of alginate
microcapsules, with highly controlled morphological and
dimensional properties, intended for cell encapsulation
and tissue engineering applications. In an attempt to
reconstitute the cell environment in a immunoisolating
device for cell immobilization, we entrapped a powder
form of ECM, isolated and purified from urinary bladder
(Urinary Bladder Matrix, UMB), together with living cells
(primary cells), in alginate based microcapsules. The aim
was to demonstrate that UBM powder can produce an
optimal substrate for in vitro culture, possibly
ameliorating the viability and functions of the coentrapped cells. In particular, the combined use of
alginate and urinary bladder matrix resulted in a synergic
activity of both materials. On one side, the engineerized
microcapsules offer the mechanical and material
properties of alginate, which can be, in addition, varied
through "on demand" gelling procedures. On the other
side, UBM provides an array of bioactive functions that
ameliorate the viability and functions of the co-entrapped
cells. Taking these features into consideration, alginate
microcapsules were applied to primary cells
encapsulation as potentially immunoprotective barrier
material and extracellular matrix (UBM) was immobilized,
into the alginate microcapsules, to promote cells survival
and function within the encapsulation microenvironment.
We demonstrated that the incorporation of UBM powder
does not alter significantly the morphological and
dimensional characteristics of the microparticles (see Fig
1) and the presence of the co-entrapped UBM promote
cell viability and function. In conclusion, the engineerized
microcapsules, here presented, may represent a novel
approach to enhance immunological acceptance and to
implement viability of the entrapped cells for tissue
Keywords. Hydrogel, urinary bladder, microcapsules
(15.O6)
FORMATION
OF
HARVESTABLE
CELL
AGGREGATES IN RESPONSIVE HYDROGEL MICROWELLS
FOR HIGH-THROUGHPUT SYSTEMS
Tekin H (1), Anaya M (1), Nauman C (1), Langer R (1),
Khademhosseini A (2)
1. Massachusetts Institute of Technology; 2. Brigham and
Women’s Hospital, Harvard Medical School
Fabrication of cell aggregates and their high-throughput
analysis are potentially useful for stem-cell engineering,
tissue engineering, and drug discovery applications. Highthroughput systems require stable aggregate formation
to prevent deformation in cell clusters under flow and
agitations and their retrieval for further use and analysis.
Soft lithographically fabricated hydrogel based microwell
structures were proven to be useful tools for aggregate
formation and integration in high-throughput systems.
Previously, glass bottomed poly(ethylene glycol)
microwell arrays were offered to stably form the cell
aggregates, though it was hard to harvest the aggregates
from these templates without using digestive enzymes or
physical forces which can potentially deform cell clusters.
In this study, we fabricated glass bottom thermoresponsive microwells to generate cell aggregates on
adhesive substrates and harvest them from microwells by
utilizing the temperature dependent swelling property of
responsive hydrogel. Temperature mediated swelling of
the responsive polymer regulated microwell shapes
applying mechanical forces on cell clusters which
subsequently allowed their ejection from microwells.
Given their ability to stably form aggregates and facilitate
their further retrieval, these thermo-responsive microwell
arrays can be potentially useful for stem cell biology,
modular tissue engineering, and drug discovery and be
applicable in high-throughput screening systems.
Keywords. Cell aggregates, responsive hydrogels,
microwell arrays, high-throughput systems
(15.O7) ENGINEERING A MSC SEEDED FIBRIN HYDROGEL
CONTAINING
TGF-BETA
1
LOADED
GELATIN
MICROSPHERES FOR CARTILAGE REPAIR
Ahearne M (1), Buckley CT (1), Kelly DJ (1)
Introduction. Articular cartilage has a limited capacity for
repair. Tissue engineering using mesenchymal stem cells
(MSC)seeded within hydrogels has been promoted as a
potential solution to repair cartilage defects. A central
challenge with such an approach is creating an
appropriate biochemical environment to allow MSCs to
undergo
chrondrogenic
differentiation
following
implantation. The objective of this study is to develop a
MSC seeded fibrin hydrogel containing TGF-β1 loaded
gelatin microspheres to allow a controlled release of
growth factor over a prolonged period.
Methods. Microspheres of diameter 50-100 µm were
manufactured and loaded with TGF-β1. MSCs derived
from porcine infrapatellar fat pad (IFP) were used for this
study. Fibrin was prepared by mixing thrombin and
fibrinogen to give a final fibrin concentration of 50mg/ml.
The microspheres and cells were suspended throughout
the fibrin hydrogel prior to gelation. The release of TGFβ1 over a 21 day culture period was measured using an
ELISA. Chondrogenesis was examined by measuring sGAG
production using Alcian blue staining and a DMMB assay
and collagen production using picro-sirius red staining
and a hydroxyproline assay.
Results. A sustained release of TGF-β1 over the 21 day
culture period was observed (Fig.1a). Release tended to
be lower in hydrogels seeded with cells than those
without cells. GAG and collagen accumulation was
significantly higher after 21 days in hydrogels containing
TGF-β1 compared to hydrogels with TGF-β1 free
microspheres (Fig. 1b).
Conclusions. It has been demonstrated that TGF-β1
loaded gelatin microspheres embedded in a fibrin
hydrogel enabled the controlled release of growth factors
capable of inducing chondrogenesis of IFP derived MSCs
and hence promoting the release and accumulation of
cartilaginous extracellular matrix components within the
hydrogel. We believe these advanced hydrogel systems
have the potential to be used for cartilage defect repair.
Acknowledgements. Funding provided by the European
Research Council.
Keywords. Hydrogel, stem cells, cartilage, growth factors
(15.O8) EVALUATION OF POLYELECTROLYTE BASED
SCAFFOLDS FOR MSCs HEART THERAPY
Ceccaldi C (1), Girod S (1), Alfarano C (2), Cussac D (2),
Parini A (2), Sallerin B (2)
1. CIRIMAT; 2. I2MC
Introduction. The aim of this work is to engineer
biocompatible materials to improve the efficiency of cell
therapy in the treatment of myocardial ischemia.
Injection in the damaged organ of mesenchymal stem
cells (MSCs) is already used as a therapeutic strategy
subsequently to infarction and reperfusion. Unfortunately
the therapeutic benefits are limited by early cell death in
the first three days after graft. A tailored scaffold, able to
improve cell survival and efficiency by providing to MSCs
a biomimetic and protective environment would be of
great interest to encapsulate and localize transplanted
cells near the injury site, promote their viability and
paracrine activity. Among all, the porosity of the scaffold
appears as a key parameter to control MCSs survival and
fate post implantation. Scaffolds three-dimensional
structure and mechanical resistance may also play a
major role in cell attachment and commitment. In this
context, generating 3-D patches based on polyelectrolyte
complexes (PEC) seems to be promising.
Materials and Methods. Ultrapur alginate and medium
Mw chitosan were used to generate patch scaffolds of
10mm diameter and 2mm thickness. Interaction between
polymers chains was studied by confocal microscopy.
Matrices were characterized in terms of microstructure,
porosity, swelling and mechanical properties. Scaffolds
with acceptable physico-chemical properties were then
loaded with human MSCs and tested in vitro. hMSCs
viability, functionality and attachment were evaluated.
Results. Scaffolds exhibiting various Chitosan / Alginate
ratios were prepared. Whatever A/C ratio, scaffolds
exhibiting an interconnected porosity (average pore size
100 µm), with maintained biocompatibility, were
obtained. Results clearly showed that chitosan addition
improved scaffolds adhesivity and mechanical properties.
Moreover, MSCs cytoskeleton organization revealed
better cell attachment.
Conclusion. This study demonstrates the interest of using
PEC to generate porous scaffolds for mesenchymal stem
cell delivery on ischemic myocardium. In vivo tests are
currently under investigation in our laboratory.
Keywords. Polymers, mesenchymal stem cells,
myocardial infarction
(15.O9) OPTIMISING MICROGEL NICHES TO INFLUENCE
MESENCHYMAL STEM CELL DIFFERENTIATION
Fontana G (1), Collin E (1), Aburub M (1), Pandit A (1)
1. Network of Excellence for Functional Biomaterials
(NFB), National University of Ireland, Galway
Introduction. Low back pain is associated with
degeneration of the intervertebral disc (IVD) and affects
the quality of life in our society. Cell therapy of the IVD is
limited by the lack of appropriate cell sources, thus
appropriate strategies for the differentiation of stem cells
to nucleus pulposus (NP) cells-like phenotype have to be
found. In the native IVD, NP cells are found sparsely in
spherical microenviroments of coll II and proteoglycans
that are known to influence the differentiation of stem
cells. It is hypothesized that, spherical niche-like
structures composed of type II collagen (coll II) hyaluronan (HA) will mimic the NP microenvironment and
promote the differentiation of adipose derived stem cells
(ADSCs) to an NP cell-like phenotype. The specific
objective of the study is to create the optimal
microenvironment to promote the differentiation of
ADSCs by varying coll II/HA concentration, cell density
and amount of crosslinking.
Material and methods. Microgels were created by mixing
coll II in different concentrations with HA at a ratio of 9:1
respectively. Cells (ADSCs or NP) were encapsulated
within the hydrogels varying their density (105-107/mL).
Different concentrations of a (ethylene glycol)-based
crosslinker were mixed to the solution with coll II/crosslinker ratios (1:1, 1:2, 1:4). The hydrogels were then
deposited on a hydrophobic surface to create a spherical
shape and incubated for 1h at 37°C. The hydrogels were
maintained in culture for 14 days before assessment of
cell viability, GAGs synthesis and gene expression.
Results. The viability of both NP cells and ADSCs is
maintained after encapsulation. The characterization of
NP cells revealed high GAGs and coll II expression. These
results show that the niche-like structure of microgels
and their composition are able to maintain the NP cells’
phenotype, and therefore this is a promising strategy for
the induction of NP-like differentiation of ADSCs.
Acknowledgement. European Commission under the
DISC REGENERATION project (NMP3-LA-2008-213904).
Keywords. Intervertebral disc, Adipose derived stem cells,
Cell delivery, niches
(15.O10) ROLE OF GLYOXALASE 1 IN DEFECTIVE
ISCHEMIA-INDUCED NEOVASCULARIZATION IN DIABETES
Vulesevic B (1), McBane J (1), Geoffrion M (1), Milne R (1),
Suuronen EJ (1)
1. University of Ottawa Heart Institute, Canada
Introduction. Vascular dysfunction caused by diabetes
leads to tissue ischemia and impaired wound healing. This
study examines possible links between the diabetic
condition, the defect in circulating progenitor cells (CPCs)
and the lack of angiogenesis. In diabetes, methylglyoxal
accumulation reduces the hypoxia-inducible factor 1dependent expression of angiogenic genes, thereby
inhibiting neovascularization. We hypothesize that this
defective neovascularization can be reversed by
increasing the activity of glyoxalase-1 (GLO1), which
metabolizes methylglyoxal.
Methods. Bone marrow (BM) cells were extracted from
control mice (C57/BL6) and mice that overexpress human
GLO1 (hGLO+), and transplanted into irradiated control
mice (+streptozotocin-induced diabetes). Hindlimb
ischemia was induced, CPC mobilization was analyzed by
flow cytometry, perfusion was analyzed by laser Doppler,
and immunohistochemistry and cytokine arrays of tissues
were performed.
Results. Compared to baseline, the number of mobilized
angiogenic CXCR4+ CPCs increased 2.4-fold in mice with
hGLO1+ BM cells at 1-day post-ischemia, versus a 1.1-fold
change in control mice (p<0.05). Total mobilization of
CPCs (2.3-fold increase) was greater in mice with hGLO1+
BM cells by day 7 compared to controls (0.9-fold; p=0.04).
The tissue level of vascular endothelial growth factor was
1.33-fold greater in mice with hGLO+ BM cells vs. control
diabetic mice. Vascular density and incorporation of CPCs
into vasculature was greater in mice with hGLO1+ BM
cells compared to wild-type mice, as determined by
staining for von Willebrand factor (endothelial cells) and
α-smooth muscle actin (arterioles). In addition, reduced
perfusion (ischemic/non-ischemic ratio) was unchanged
in control mice after 2 weeks (49±4%), but was restored
in mice with hGLO+ BM cells (84±13%; p=0.02).
Conclusion. This evidence suggests that GLO1 is a
potential target to restore CPC function and
neovascularization in diabetes.
Keywords. Glyoxalase-1, diabetes, hindlimb ischemia,
neovascularization
(15.O11)
MULTI-MATERIAL
PRINTING
FOR
HETEROGENEOUS TISSUE SCAFFOLDS
Koc B (1), Ozbolat IT (2)
1. Sabanci University; 2. University of Iowa
Introduction. Alginates have been widely applied as
hydrogel synthetic extracellular matrices (ECMs) in
wound care due to their gelatin property during in
contact with body fluid. Due to short biological half life,
potential carcinogenesis risk and lack of tissue selectivity,
release kinetics of proteins and growth factors needs to
be controlled temporarily and spatially. This research
aims to develop heterogeneous wound scaffolds with
localized control of release kintics of active materials.
Pressure assisted multi-chamber single nozzle deposition
system is used to fabricate wound scaffolds with multimaterial.
Materials and Methods. Sodium alginate from brown
algae and calcium chloride were purchased from SigmaAldrich. Nozzle tips for dispensing systems were
purchased from EFD. 3%- 4.5% (w/v) alginate solutions
with different concentration and colors were prepared
and loaded into Chamber A and Chamber B in the
fabrication unit respectively. Solutions were deposited
through multi-chamber single nozzle dispensing system
with 250 µm nozzle tip. Calcium chloride solution with
0.6% (w/v) DI water then dispensed onto printed alginate
structure through another nozzle for crosslinking
purpose.
Results. Wound image of a pressure ulcer from [5] is
processed in Image J software (See Fig. 1(a)). The wound
geometry is then inputted into feature-based 3D blending
process to generate heterogeneous wound scaffolds with
uniform regions (in Fig. 1(b)). In Fig. 1(c), a concentration
profile is shown as a continuous function increasing from
3% to 4.5% assumed to follow tissue engineering and
wound healing needs. Finally, heterogeneous scaffold
shown in Fig. 1(d) is printed with four regions.
Discussion and Conclusions. Heterogeneous wound
scaffolds with varying material concentration is designed
and fabricated in a way that wound healing process can
be synchronized with release kinetics of any loaded
proteins.
Acknowledgement. This research is supported partially
by DoD, U.S. Army Medical Research Grant #: W81XWH05-1-0401.
References.
1. Ribeiro C. Biomaterials. 2004;25(18):4363-4373.
2. Juliano R.L. J Cell Biol.1993;120:557-585.
3. Putney SD. Nature Biotecnol.1998;16:153-157.
4. Perkins J. Int. Mec. Eng. Congress & Expo.,FL,2009.
5. Albouy B. 29th Ann. Int. Con. of IEEE, France, 2007.
6. Ozbolat IT. IERC, Mexico, 2010.
Keywords. Scaffold printing, alginate, heterogenous
scaffold
(15.O12) CONTROLLED RELEASE OF STROMAL CELLDERIVED FACTOR-1 FOR ENHANCED PROGENITOR CELL
RESPONSES IN ISCHEMIA
Kuraitis D (1), Zhang P (1), McEwan K (1), Sofrenovic T (1),
Zhang Y (1), McKee D (1), Zhang J (2), Griffith M (2), Cao X
(2), Ruel M (1), Suuronen EJ (1)
1. University of Ottawa Heart Institute; 2. University of
Ottawa
Introduction. Following an ischemic event, the body
releases stromal cell-derived factor-1 (SDF-1) in an effort
to recruit CXCR4+ circulating progenitor cells (CPCs) to
injured sites; but this is insufficient for effective repair.
The current study aims to enhance this endogenous
response by injecting a collagen matrix with SDF-1
releasing microspheres into ischemic muscle.
Methods. Alginate microspheres (+/- SDF-1) were created
using a spray gun/air compressor. Matrix was created by
blending collagen I and chondroitin sulfate on ice, and
cross-linking with EDC/NHS. CPCs were isolated from
healthy human donors, and cultured in the presence of
blank or SDF-1-loaded microspheres. Rheology and SDF-1
release was assessed for matrices +/- SDF-1
microspheres. Femoral arteries of mice were ligated, and
animals received intramuscular injections of: PBS, matrix,
or SDF-1-matrix. CPCs and hindlimb perfusion were
assessed over 2 weeks. After sacrifice, hindlimbs were
assessed for arterioles, CXCR4+ CPC engraftment, and
cytokine profiles.
Results. Adding microspheres increased matrix viscosity
by 17%, and prolonged SDF-1 release from approximately
1 to 10 days. SDF-1 microspheres were bioactive; 2.3- and
3.2-fold more CPCs were adhesive and migrative in their
presence, respectively, compared to blank microspheres.
From days 1-14 post-ligation, SDF-1-matrix treatment
increased flk+ CPCs; and earlier and later time points saw
respective increases in CXCR4+ and c-kit+ CPCs. At 14
days, matrix and SDF-1-matrix treatments restored
hindlimb perfusion, and SDF-1 treatment increased
arteriole size ≥2
by
-fold. Matrix and SDF-1-matrix
treatments recruited 2.5- and 4.5-fold more CXCR4+ cells,
respectively, compared to PBS. SDF-1-matrix treatment
also reduced inflammatory cytokines IL-1α, MIP-3α, and
increased angiogenic cytokines IGF-1, bFGF. For all
results, p<0.05.
Conclusions. We have exploited the SDF-1 axis to increase
CPC activity following an ischemic event. Injection of a
matrix with SDF-1 microspheres allows for controlled SDF1 release, recruitment of CPCs, increased vascularity, and
restoration of perfusion.
Keywords. Angiogenesis, Circulating Progenitor Cell,
Ischemia, Stromal Cell-Derived Factor-1
(15.O13) MULTIGRADIENT HYDROGELS TO DECODE
EXTRINSIC REGULATION OF HEMATOPOIETIC STEM CELL
FATE
Mahadik B (1), Wheeler TD (1), Kenis PJAK (1), Harley BA
(1)
1. Dept. of Chemical and Biomolecular Engineering,
University of Illinois at Urbana Champaign, USA
Introduction. Hematopoietic stem cells (HSCs) are
responsible for the generation of all blood and immune
cells of the body. HSCs are primarily found in specific
microenvironments (niches) within the bone marrow. The
HSC niche, composed of other cell types, the ECM and
soluble biomolecules, is thought to provide extrinsic
signals that influence HSC fate decisions. However, little is
known about the mechanisms that underlie niche
regulation. Here, we develop novel 3D biomaterial
systems that mimic aspects of the complex niche
microenvironment in order to systematically assess the
influence of cell-cell interactions on HSC fate.
Methods. We create multiple opposing gradients of cells
and/or hydrogel biomaterials in a novel multi-gradient
microfluidic chamber (~180 uL volume) to encapsulate
HSCs and Osteoblasts (a putative niche cell) in a collagen
hydrogel. The HSCs are isolated as Lin-c-kit+Sca-1+ from
murine bone marrow. Discrete sections within this
chamber contain defined ratios of HSC:niche cells that
can be isolated to probe HSC biology using tools such as
surface antigen expression, MTS assay, gene expression
and functional assays.
Results. We have successfully created opposing gradients
of fluorescent microbeads (1 µm dia. FluoSpheres,
Invitrogen), osteoblasts and HSCs:osteoblasts (Figure A) in
distinct collagen suspensions (1 – 2.5 mg/mL). We have
used fluorescent image scanning as well as flow
cytometry and imaging of discrete regions to quantify the
resultant gradients (Figure B). We hypothesize that comodulating the local niche cell and hydrogel densities
while applying known biomolecules of the HSC signaling
cascades will enable us to understand and quantify direct
vs. indirect (paracrine signaling) interactions between
niche cells and HSCs.
Conclusion. This project will develop transformative, high
throughput tools to systematically explore the
significance of cell-based cues on HSC fate and provide
significant new insight into the relationship between
extrinsic cues and internal signaling cascades regulating
HSC biology.
Keywords. Hematopoietic, gradient, biomaterial
(15.O14) IN VIVO EVALUATION OF ANGIOGENIC
FACTORS IN A COLLAGEN-CHITOSAN MATRIX AS A
POTENTIAL ISLET TRANSPLANT SITE
McBane JE (1), Vulesevic B (1), Ellis C (2), Korbutt G (2),
Suuronen EJ (1)
1. University of Ottawa Heart Institute, Ottawa, Canada;
2. University of Alberta, Edmonton, Canada
Introduction. Islet transplantation for the treatment of
type I diabetes often fails due to a lack of proper blood
supply to support islet survival at the transplant site.
Endothelial progenitor cells (EPCs) promote angiogenesis
while collagen matrices can promote the homing of
functional EPCs. Adding chitosan to these collagen
matrices improves matrix stability and enhances their
ability to stimulate angiogenesis in vitro. In vivo data
suggests that collagen-chitosan matrices better stimulate
vascular growth;1 however, the mechanism(s)
responsible, such as the expression of pro-angiogenic
growth factors were not evaluated. In the current study,
collagen and collagen-chitosan matrices +/- EPCs were
tested for their ability to promote pro-angiogenic
cytokines in vivo and viability of islets cultured in vitro.
Methods. Human peripheral blood mononuclear cells
were seeded onto fibronectin-coated tissue culture
polystyrene for 4d to select for EPCs. Collagen and 10:1
collagen:chitosan matrices +/- EPCs were subcutaneously
implanted into the backs of nude mice (n=4). After 14d,
the explants were analyzed using RayBiotech® Mouse
(Angiogenesis) Cytokine arrays. Neonatal pig islets were
harvested from the pancreas and cultured in islet media,
collagen or collagen-chitosan matrices for up to 7d.
Results. Both matrices promoted cell infiltration. Twentyone pro-angiogenic cytokines were significantly
stimulated in the collagen-chitosan matrix compared to
collagen matrices (+/- EPCs; p<0.05) including VEGF which
has been shown to be important for promoting islet
vascularization and function post-transplantation.2 Proangiogenic factors monocyte chemotactic protein-5,
eotaxin and keratinocyte chemoattractant were also
stimulated. In vitro, neonatal islets in the collagenchitosan matrix showed similar responses to controls.
Conclusion. The collagen-chitosan matrix promotes
production/retention of pro-angiogenic cytokines
compared to collagen-only matrix, which may contribute
to the increased vascularization observed in vivo using
these matrices. Therefore, the collagen-chitosan matrix
warrants further evaluation in islet transplantation
models.
References.
1. Deng. Tissue Eng Part A, 2010; 16:3099.
2. Brissova. Diabetes, 2006; 55:2974.
Keywords. Collagen, chitosan, EPC, islet, diabetes
(15.O15)
MICRO-ENGINEERING
VASCULAR-LIKE
STRUCTURES BY MEANS OF ELECTROCHEMICAL CELL
PRINTING IN PHOTO-CROSSLINKABLE HYDROGELS
Sadr N (1,2,3), Zhu M (4), Osaki T (5), Kakegawa T (5),
Moretti M (6), Fukuda J (5), Khademhosseini A (1,2,3)
1. Harvard-MIT Division of Health Sciences and
Technology, Massachusetts Institute of Technology,
Cambridge, MA; 2. Center for Biomedical Engineering,
Department of Medicine, Brigham and Women’s Hospital,
Harvard Medical School, Cambridge, MA; 3. Wyss Institute
for Biologically Inspired Engineering, Harvard University,
Boston, MA; 4. Department of Biology, Mount Holyoke
College, USA; 5. Graduate School of Pure and Applied
Sciences, University of Tsukuba, Japan; 6. Cell and Tissue
Engineering Lab, IRCCS Istituto Ortopedico Galeazzi, Italy
Introduction. A key challenge in engineering functional
tissues in vitro is reproducing the complex in vivo 3D
micro-architecture, in particular the controlled generation
of vascular networks. In this work we propose and
characterize a new tissue micro-engineering approach,
relying on the combination of electrical cell printing and
hydrogel photo-patterning techniques, to fabricate
vascular-like structures.
Materials and Methods. Human umbilical vein
endothelial cells (HUVECs) were seeded on gold
substrates previously modified with self-assembled
monolayers (SAM) of an RGD containing oligopeptide via
electrically cleavable gold-thiolate bonds. Cell transfer to
photo-crosslinkable gelatin methacrylate (GelMA)
hydrogels was then investigated, w or w/o electrical
potential application, at 16/72 h of culture. Vascular-like
structures were generated by culturing HUVECs on SAMmodified gold rods and transferring the resulting tubular
monolayers to the inner surfaces of micro-channels in
plain or cell laden GelMA hydrogels.
Results. Cell transfer efficiency to GelMA at 16 h of
culture, both w/ and w/o electrical potential application,
was close to 100%. At 72 h of culture, cell transfer w/o
potential application was decreased to ~30%, whereas
electrical potential application improved it to ~86% and
allowed to maintain VE-Cadherin cell-cell interactions in
transferred HUVEC monolayers .
With this approach 600-µm-diameter capillary-like
structures were fabricated in hydrogels, which remained
persistent in perfusion culture for at least 15 d. As a first
step toward more complex and in vivo-like structures an
additional cell laden hydrogel layer was photo-patterned
around the HUVEC channel mimicking the vascular
smooth muscle cell layers.
Conclusions. Our results show that coupling electrical cell
printing and photo-patternable GelMA hydrogels is a fast
and reliable method with great potential for devising
micro-engineered endothelialized 3D vasculature for
regenerative medicine or in vitro drug screening
applications.
Acknowledgments. Authors acknowledge support
provided by the NIH, NSF and by the Progetto Rocca.
Keywords. Oligopeptide self-assembled monolayers,
methacrylated gelatin tissue engineering
(15.O16)
INNOVATIVE
CROSSLINKED
GELATIN
HYDROGELS AS KEY SCAFFOLDS FOR ADIPOSE STEM
CELLS DIFFERENTIATION
Faré S (1), Gerges I (1), D'Ercole E (1), Altomare L (1),
Tanzi MC (1)
1. Politecnico di Milano
Introduction. Gelatin-based hydrogels may find
application in drug delivery, wound dressing and as
scaffolds for tissue regeneration. The aim of this work
was to design hydrogels in which gelatin was covalently
crosslinked with a synthetic component, so to conjugate
the ability of gelatin to promote cellular adhesion with an
adequate mechanical stability.
Materials and Methods. Gelatin A (from bovine skin,
Sigma) was crosslinked by Michael-type addition with
methylene-bis-acrylamide (MBA). The efficiency of the
reactions was evaluated with FTIR spectroscopy. Swelling
and weight loss were studied in distilled water and
phosphate buffered saline (PBS, pH = 7.4) at 37°C.
Mechanical properties of the swollen samples were
assessed in an unconfined cyclic frequency sweep and
stress relaxation-recovery compression tests. Cytotoxicity
was evaluated in vitro by an indirect contact test with
L929 fibroblasts and cell viability of adipose mesenchimal
stem cells (provided by Istituto Nazionale dei Tumori,
Milano, I) cultured onto the hydrogels for 7 days was
assessed by optical microscopy and MTT assay and their
differentiation into adipocytes was examined by oil red O
staining.
Results and Discussion. All the synthesized hydrogels
were stable in distilled water and PBS up to 35 days. The
hydrogels exhibited E’ values in the range 2.5 – 23 kPa.
Stress relaxation-recovery tests evidenced the
predominance of elastic behavior on the viscous one. No
release of cytotoxic degradation products was detected
up to 7 days with L929 cells. Oil red O staining showed a
differentiation of ASCs in adipocytes onto all the prepared
hydrogels (Fig.1).
Keywords. Gelatin hydrogels, mechanical properties,
adipose tissue regeneration
a)
b)
Figure 1 – Oil red O staining of ASCs differentiated into
adipocytes onto two gelatin/MBA hydrogels
(15.O17) SELF-GENERATED CONSUMPTION GRADIENTS
BY STEM CELLS IN 3D DETERMINE ANGIOGENIC
SIGNALLING
Cheema U (1), Mudera V (1)
1. UCL Tissue Repair and Engineering Centre, Division of
surgery and interventional sciences, RNOH Stanmore
campus, London, HA7 4LP, UK
The specific O2 environment in which cells reside affects
their phenotype and signalling. Stem cells in the bone
marrow cavity typically reside in O2 tensions of between
1-5%. Most in vitro cell culture is done in O2 tensions of
around 21%, which can translate as oxidative stress to a
cell. By culturing cells within 3D, we have previously
reported O2 consumption gradients from the surface to
the core, ranging from between 18-3%1. We have now
tested the effect of O2 gradients (3-18%) generated by
stem cells within these tissue models and its effect on
generating the angiogenic signalling cascade in distinct
locations with 3D constructs exposed to specific O2 levels.
We found that vascular endothelial growth factor (VEGF)
expression was up regulated earlier in core cells (3% O2)
(significantly increased by day 4) compared to surface
cells (18%O2), where no significant increase in VEGF was
measured up to 6 days (figure 1). This pattern was also
similar for Hypoxia-Inducible Factor Iα (HIF-Iα) and
Platelet derived growth factor (PDGF), both critical factors
in the angiogenic cascade. Low O2 did not have an effect
on cell viability tested up to 6 days. We therefore
conclude that Stem cells when, exposed to 3-5% O2
optimally up-regulated the three angiogenic factors
tested within 4 days, whilst still maintaining cell viability.
In stem cell based therapies, where angiogenic response
is critical (e.g. non-union fractures where stem cell
therapies are being tested and delayed fracture healing
where angiogenic response is poor) using hypoxia to
generate these angiogenic cascade signals in vitro prior to
in vivo implantation has potential therapeutic benefits.
References: 1. Cheema, U. Brown, R.A. Alp, B. MacRobert,
A.J. (2008). ‘Spatially defined oxygen gradients and VEGF
expression in an engineered 3D cell model.’ Cell. Mol. Life
Sci. 65(1): 177-186
Keywords. Stem Cells, Collagen type I scaffolds
5-7%
3-5%
~18%
Cell-seeded construct
Figure 1. O2 gradient formation in spatially distinct regions of
seeded construct, with VEGF signalling over 6 days.
(15.P1)
STUDY
OF
BIOMATERIAL-HEPATOCYTE
CONSTRUCTS: A FUNDAMENTAL BASIS FOR THE
DEVELOPMENT OF BIO-ARTIFICIAL LIVER DEVICES
Gevaert E (1), Billiet T (1), Vandenhaute M (1), Dubruel P
(1), Cornelissen M (1)
1. Ghent University
Introduction. Artificial liver support systems are urgently
needed as bridge to liver transplantation or regeneration.
The search for a bio-artificial system is hampered by the
difficulty to keep hepatocytes fully functional and
differentiated, once isolated from the liver. The answer to
this problem might lay in the biomaterial that is used for
cultivation or encapsulation of the cells. Our goal is to
compare different biomaterials, modifications and cellpacking methods, and to select a material and set of
conditions that is biocompatible and allows hepatocytes
to maintain full differentiation.
Methods. HepG2 cells are encapsulated in different
biomaterials and viability is followed over time via
standardized viability assays such as MTT assay,
calceïne/propidium iodide staining. In case of acceptable
viability, hepatocyte phenotype is checked via
immunohistochemistry stainings, collimetric assays and
ELISA techniques. Based on the results, the biomaterials
are evaluated and adapted if necessary.
Results and conclusions. Different equivalents and w/v%
of metacrylate-modified gelatin were, and are currently,
tested. Until now, the gelatin-metacrylate (1 eq., 10
w/v%) shows the best results for the encapsulation of
HepG2 cells with a viability of about 70 %, compared to
the control culture. The encapsulated cells still show
storage of glycogen, expression of HNF4α and albumin
but, in general, less than the control culture. Currently we
are investigating whether additional modifications with
galactose could improve the phenotype. When
encapsulated in bismetacrylate-modified pluronic F127,
calceïne/PI staining of the HepG2 cells suggests problems
with membrane integrity, while mainly death cells were
seen when cells were encapsulated in bisacrylatemodified pluronic F127. When cells were encapsulated in
pluronic-ALA-L , mainly living cells were observed. The
current results show that small changes to biomaterial
parameters have a great influence on the cell behavior
and that it might be possible to find conditions where the
cells maintain full differentiation.
Keywords. Gelatin, pluronic, hepatocytes
(15.P2) TRABECULAR TITANIUM™ COMBINED WITH A
CELLULOSE-BASED HYDROGEL AND HUMAN BONE
MARROW STROMAL CELLS: AN INNOVATIVE STRATEGY
TO IMPROVE IMPLANT OSTEOINTEGRATION
Lopa S (1), Tavola M (1), Pedroli S (1), Mercuri D (2),
Segatti F (3), Zagra L (1), Moretti M (1)
1. Cell and Tissue Engineering Lab, IRCCS Galeazzi
Orthopaedic Institute, Milan, Italy; 2. BioSuMa srl,
Villanova di San Daniele, Udine, Italy; 3. LIMA Corporate
spa, Villanova di San Daniele, Udine, Italy
Introduction. Titanium is widely used for several medical
implants but the interface between implant and bone
remains the weakest point during the initial healing
period. To accelerate osteointegration we propose to use
an amidated carboxymethylcellulose hydrogel (CMCA)
seeded with bone marrow stromal cells (BMSCs)
combined with Trabecular Titanium™, a material
characterized by an innovative multiplanar hexagonal cell
structure imitating trabecular bone. To validate this
composite bioconstruct we determined BMSCs viability
and osteogenic differentiation on CMCA and on
Trabecular Titanium™ disks unloaded (TT) or loaded with
CMCA (TT+CMCA, Fig.1a).
Methods. Human BMSCs were harvested from 9 donors
(59±2 years), under written consent. BMSCs were
expanded, seeded on CMCA, TT and TT+CMCA (7.5x10^5
cells/sample) and cultured in control (CTRL) or osteogenic
(OSTEO) medium. After 7, 14 and 21 days on biomaterials,
viability and proliferation of BMSCs was determined and
alkaline phosphatase activity (ALP) was measured to
evaluate osteogenic differentiation.
Results. CMCA well supported BMSCs growth during the
whole culture period, with a significantly higher viability
in OSTEO BMSCs compared to CTRL BMSCs up to 14 days
of culture. After 14 and 21 days on CMCA, OSTEO BMSCs
showed higher levels of ALP compared to CTRL BMSCs,
demonstrating that culture on CMCA allowed cells to
maintain their osteogenic potential. OSTEO BMSCs were
able to grow on TT and TT+CMCA, maintaining a high
viability for the entire culture. The presence of CMCA
ameliorated the osteo-conductive properties of TT as
shown by increased ALP levels (+61%) after 21 days of
culture for BMSCs on TT+CMCA compared to BMSCs on
TT (Fig.1b).
Conclusions. Based on these results, the use of a
composite construct as TT+CMCA represents a promising
approach to deliver and retain autologous BMSCs at the
implant site and to accelerate osteointegration.
Acknowledgements. This work was supported by Italian
Ministry of Health (Project RF-IOG-2007-647233).
Keywords. Titanium, Stem cells, Hydrogel, Bone.
Figure 1: (a) Section of TT+CMCA colored with azorubine
(CMCA stained in red); (b) ALP activity in OSTEO BMSCs
cultured for 21 days on TT and TT+CMCA (ALP Units/µg of
protein).
(15.P3) POTENTIAL OF A NEW TYPE OF LOW MOLECULAR
WEIGHT HYDROGEL FOR IN VITRO CULTURE AND IN
VIVO IMPLANTATION OF HUMAN ADULT STEM CELLS
Ziane S (1), Patwa A (2), Barthélémy P (2), Chassande O
(1)
1. INSERM U1026; 2. INSERM U869
A new thermosensitive hydrogel based on low molecular
weight Glycosyl-nucleoside-lipid (GNF) Units, has been
recently synthesized via double and simple click chemistry
approaches. This hydrogel is characterized by a selfassembly of amphiphile monomers which constitute a
network of nanofibers in an aqueous environment. This
hydrogel appears as a potential scaffold for cell culture
and tissue engineering applications. Indeed, the
incorporation of a fluorocarboned moiety as the
hydrophobic part of the GNF has been shown to improve
the cytocompatibility of this hydrogel. We investigated
the possibility to use GNF hydrogels as a scaffold for
adipose tissue stem cells (ADSCs) for in vitro culture and
in vivo implantation. ADSCs are pluripotent mesenchymal
stem cells isolated from adipose tissue and capable of
differentiating into several cell types.
In vitro and in vivo assays showed the high stability of the
GNF gel. Indeed, when implanted subcutaneously, GNF
hydrogel was still present after four weeks. GNF hydrogel
was then tested as culture support for human ADSCs.
Cytotoxicity assays showed a good cytocompatibility.
When isolated ADSCs were seeded either on the surface
or within the GNF hydrogels, they were unable to adhere
and spread normally, resulting in cell death after a few
days. However, ADSCs cultured as spheroids trapped in
the GNF gel demonstrated a long term survival of cells
and stability of the spheroids. When ADSCs spheroids
encapsulated in GNF hydrogels were implanted
subcutaneously in mice, cells were shown to survive and
to remain as clusters after at least one month. In contrast,
spheroids implanted without gel were far less stable. In
addition, the hydrogel was colonized by host cells and
features of inflammation were observed.
Together, these data are encouraging to propose GNF
hydrogels as a scaffold for tissue engineering and
regenerative medicine applications.
Keywords. Stem cells, thermosensitive hydrogel
(15.P4) INFLUENCE OF BIOFUNCTIONALIZED PEPMHA
HYDROGELS
ON
MESENCHYMAL
STEM
CELL
Magalhaes J (1), Ruiz OMI (1), Blanco FJ (1), Roman JS (2)
1. INIBIC; 2. ICTP, CSIC
Introduction. The process of chondrogenesis within a
healing articular cartilage lesion can be enhanced by
regenerative signals provided to the site of articular
cartilage repair. Several growth factors, such as the family
of transforming growth factors-βs (TGF-βs), have shown
to play an important role in the growth and
differentiation of articular cartilage as well as in the
chondrogenic differentiation of mesenchymal stem cells
(MSCs). Semi-interpenetrated networks (SIPNs) of poly[2ethyl(2-pyrrolidone) methacrylate] (PEPM) and hyaluronic
acid (HA)- PEPMHA, are biocompatible and can be
potential candidates as delivery vehicles for bioactive
proteins. The presence of HA within this system could
provide a favourable niche for MSCs chondrogenesis, as
these cells express CD44, one of the primary receptors for
HA. We investigated the use of PEPMHA biofunctionalized
with TGF-β3 in the induction of MSCs chondrogenesis.
Methods. PEPMHA SIPNs were produced, using
triethylene glycol dimethacrylate as crosslinker and
K2S2O8 as initiator. Transforming growth factor-β3 was
directly loaded into the PEPMHA hydrogels. Effects of
growth factor release from the PEPMHA polymeric
systems on the differentiation of the mesenchymal stem
cells isolated from bone marrow stroma, previously
expanded, were studied for different periods of time. The
cell distribution, morphology, differentiation and
extracellular matrix components deposition were
determined by histological and immunohistochemical
stainings.
Results. Highly swellable PEPMHA hydrogels present a
porous structure that can be intimately related to their
swelling character, endearing themselves as highly
diffusible systems. After 21 days in culture, MSCs were
able to proliferate and secrete an extracellular matrix
incorporating type-II collagen and proteoglycans.
Conclusion. Biofunctionalization of PEPMHA hydrogels
with TGF-β3 may constitute a valid alternative for
pursuing future cartilage tissue engineering strategies by
enabling the modulation of a chondrogenic
differentiation process.
Acknowledgements. The authors would like to
acknowledge CIBER-BBN network and MICINN-PLAN E, for
providing financial support and BIOIBERICA for HA supply.
Keywords. Hyaluronic Acid, Bone Marrow derived
mesenchymal stem cells, growth factors release
(15.P5) HYPOXIA IS INFLUENCED BY CYCLIC MECHANICAL
LOADING IN EARLY FRACTURE HEALING – AN IN VITRO
ANALYSIS
Witt F (1), Petersen A (1), Duda GN (1)
1. Julius Wolff Institute, Charité – Universitätsmedizin
Berlin; Center for Musculoskeletal Surgery, Charité –
Universitätsmedizin Berlin, Germany
Introduction. Mechanical loading after fracture has an
influence on the healing outcome. The fracture damages
the vascular system and impairs the oxygen supply in the
fracture hematoma. With the lack of oxygen a hypoxic
environment develops which may lead to a delayed
healing. The aim of this study was to analyze the
influence of cyclic mechanical loading on the oxygen
tension with an in vitro setup.
Methods. Human mesenchymal stem cells were
embedded in fibrin hydrogel constructs mimicking the
fracture hematoma and placed in a bioreactor system.
Cyclic loading, adjusted to the one experienced during a
patients gait, were applied to an in vitro culture system.
The hypoxic volume inside the constructs was visualized
by immune histological staining based on a hypoxic
marker applied to the culture medium. Additionally,
oxygen tension was quantified by an optic-chemical fibersensor placed in the center of the cell construct.
Results. It was observed that mechanical stimulation has
a positive effect on the oxygen concentration in the fibrin
construct. Cyclic compressed constructs qualitatively
exhibited in histological staining a smaller hypoxic region
compared to not stimulated controls. This observation
was confirmed by quantitative data recorded with an
oxygen sensor. The oxygen tension increased in the
construct with increasing duration of cyclic compression.
Once stimulation was stopped, the oxygen tension
decreased to the value of the unloaded constructs.
Conclusions. In the early fracture healing phase, hypoxia
develops due to rupture of blood supply. Hypoxia is
important for triggering angiogenesis, however,
prolonged hypoxia might delay regeneration processes.
Diffusion-based oxygen transport is limited. However, our
investigations showed that an appropriate mechanical
loading is able to actively transport oxygen into nonvascularized tissues and expand the supplied region.
Acknowledgements. We acknowledge financial support
by Synthes GmbH.
Keywords. Oxygen tension, fibrin hydrogel, mechanical
stimulation
(15.P6)
BONE
DEVELOPMENT
WITH
HUMAN
HEMATOPOIETIC CELLS SEEDED ONTO A NATURAL
POLYMER
Sesman A (1), Ruvalcaba E (1), Lecona H (2), Baena L (3),
Solís L (4), Sánchez-Guerrero S (5), Ibarra C (1), Jiménez C
(1), Velasquillo C (1)
Medicine Unit, National Institute of Rehabilitation. INP; 2.
Bioterio, National Institute of Rehabilitation; 3.
Department of Pathology, National Institute of
Rehabilitation; 4. Electron Microscopy Unit, National
Institute of Rehabilitation; 5. Instituto Nacional de
Cancerología
Introduction. Reconstruction of craniofacial bone defects
caused by trauma and ablative oncologic procedures, or
by congenital anomalies, is a frequent surgical challenge.
Objetive. This study evaluated the feasibility of creating
tissue-engineered bone using an osteogenic unit with
human stem cells (HSC) and demineralized bone seeded
onto a dermis acellular as a scaffold.
Material and Methods. For the experimental group,
critical bone defects were made surgically in the skull of
athymic mice and repaired with the osteogenic unit in
contact with the dura mater. The control group did not
receive the osteogenic unit. Subjects were sacrificed at
three months after implantation, the neotissue was
morphologically and histologically evaluated using
descriptive histology. Also, immunohistochemical analysis
was performed to determine the presence of alkaline
phosphatase, collagen I, osteopontin and osteocalcin.
Mineral analysis was done using edax in order to evaluate
the presence of the bone main minerals.
Results. In the control group no obvious bone formation
was found. The tissue layer that covered the repaired
defect in the experimental group with the osteogenic unit
showed new bone tissue when analyzed with descriptive
histology. There was a significant increase in the
expression of phosphatase alkaline, collagen I, osteocalcin
and osteopontin in the experimental group. Likewise, the
amount of calcium, magnesium and phosphorous was
statistically significant in animals implanted with the
osteogenic unit, compared with the control group.
Conclusion. The grafts obtained in vivo through tissue
engineering using adult stem cells seeded onto dermis
acelullar, and demineralized bone, showed osteogenic
properties with potential for many clinical applications.
Acknowledgments. This study was partially supported by
a grant of CONACYT sectoriales 114359.
Keywords. Bone, tissue engineering, craniofacial defects,
scaffold
(15.P7)
GALACTOSYLATED
CELLULOSIC
SPONGE
ACCELERATES HEPATOCYTE REPOLARIZATION
Nugraha B (1), Yu H (2)
1. Institute of Bioengineering and Nanotechnology
A*STAR Singapore; 2. Yong Loo Lin School of Medicine
National University of Singapore
Hepatocytes lose their differentiated functions rapidly
upon isolation from their native niche in liver. One way to
solve this problem is by achieving rapid hepatocyte
repolarization in vitro. Culturing hepatocyte as 3D
spheroid is one approach to induce hepatocyte
repolarization due to increase in cell-cell contact.
However, many current platforms are still taking time to
repolarize (between 48-72 hours). Here we decipher our
method by using hydrogel-based sponge conjugated with
galactose, which is known as hepatocyte chemical cues in
forming spheroids, to rapidly aggregate hepatocyte upon
seeding thus accelerating functional hepatocyte polarity
formation.
Cellulosic sponges were synthesized from hydroxypropyl
cellulose (MW 10,000), which was side chain-modified
with allyl isocyanate and conjugated with β-galactose.
The obtained product was then dissolved in water and
gamma irradiated to crosslink the sponges.
Hepatocytes seeded in the sponge were monitored in
terms of their aggregation process, polarity formation
and cellular morphology.
Rat hepatocytes seeded in cellulosic sponge reorganize
themselves to form 3D spheroids within 24 hours (figure
1b). Hepatocyte aggregation itself was observed as fast as
7 hours postseeding (figure 1a), while repolarization
started at 16 hours postseeding as indicated by
accumulation of fluorescein diacetate dyes in the regions
between two neighboring cells (which is known to be bile
canaliculi, figure 1d-f). The accumulation of the dyes at 48
hours shows stronger signal, revealing more functioning
bile canaliculi. SEM images of hepatocyte spheroids after
24 hours postseeding decipher tightness of spheroids
surface and inability to distinctively present the single cell
boundary (figure 1c). This phenomena show the
robustness of our cellulosic sponge to help in inducing
hepatocye spheroids formations and restoring the
hepatocyte repolarization quickly upon seeding. Other
hydrogel to culture hepatocytes show improved
hepatocyte differentiated functions maintenance but not
readily induce rapid repolarization, which is important
factor in preserving the differentiated functions.
Keywords. Hepatocyte polarity, scaffold, hydrogel
(15.P8) CONTROLLED RELEASE BY BIODEGRADABLE
HYDROGELS (PLGA-PEG-PLGA TRIBLOCK COPOLYMER)
ENHANCES THE BONE FORMATION OF BONE
MORPHOGENETIC PROTEIN-2 ON CRITICAL-SIZE BONE
DEFECT
Chen CY (1), Li PS (2), Hsieh MY (2), Shen HH (2), Lin FH
(1), Sun JS (3)
1. Department of Biomedical Engineering, National
Taiwan University, Taipei, Taiwan, ROC; 2. Biomedical
Technology and Device Research Laboratories, Industrial
Technology Research Institute, Chutung, Hsinchu, Taiwan,
ROC; 3. Department of Orthopedic Surgery, National
Taiwan University Hospital, Taipei, Taiwan, ROC
Background and Purpose. PLGA-PEG-PLGA triblock
copolymer is a kind of thermal-sensitive hydrogel. In this
study, we used this triblock copolymer as a drug carrier
for control release. The recombinant human bone
morphogenetic protein-2 (rhBMP-2), which was
manufactured from Industrial Technology Research
Institute, plays an important role in bone formation. The
aim of this study is to prove that rhBMP-2 wrapped up in
PLGA-PEG-PLGA hydrogel and trapped in type I collagen
sponges have clinical potential on critical-size defect
model.
Methods. 45 Wistar rats were assigned into five groups
(n=9), and a 5 mm × 5 mm × 5 mm segmental bone defect
in the ulnar shafts were created by surgery. The two
experimental groups of type I collagen and type I collagen
/ PLGA-PEG-PLGA scaffolds received local injection of 8 µg
rhBMP-2. The negative control groups received type I
collagen and type I collagen / PLGA-PEG-PLGA scaffolds
soaked with PBS only. Radiographic evaluation and
histological stains were performed on bone healing and
bone formation.
Main Results. The X-ray data indicated that rhBMP-2
directly guiding bone formation at the defect site.
Through the micro-CT analysis, the data showed the
percentage of bone volume (BV/TV) significantly
increased in the experimental group, which implanted
type Ⅰ collagen / PLGA-PEG-PLGA scaffolds / rhBMP-2
scaffolds at 4, 8 and 12 weeks.
Conclusions. Through this study, we demonstrated that
the efficacy of type Ⅰ collagen / PLGA-PEG-PLGA
scaffolds combining with rhBMP-2 has better
osteoinductive effect and can promote fracture healing
on radiographs and histological stains of an experimental
critical-sized bone defect model. The results indicated
that type Ⅰ collagen / PLGA-PEG-PLGA scaffolds /
rhBMP-2 implants would provide the possible clinical
applications in orthopedic surgery and regenerative
medicine.
Keywords. PLGA-PEG-PLGA, bone morphogenic protein-2
(BMP-2), critical-size bone defect, bone healing
(15.P9)
THERMORESPONSIVE
POLY(NVINYLCAPROLACTAM)-g-COLLAGEN:
SYNTHESIS,
CHARACTERIZATION, IN VITRO CYTOTOXICITY AND IN
VIVO BIOCOMPATIBILITY EVALUATION
Durkut S (1), Elcin YM (1)
Science, TEBNL, Ankara, Turkey
Introduction. Thermoresponsive polymers have a variety
of applications in medicine and biotechnology. They are
characterized by sudden reversible phase separation with
an on-off switch using the transition between the
extended and coiled forms of the polymer brushes in
response to a certain temperature range. Poly(Nvinylcaprolactam) (PNVCL) is a non-toxic synthetic
polymer with a LCST of ~32oC. Here, we synthesized
PNVCL-g-collagen and PNVCL-g-chitosan, and evaluated
some of the physical, chemical and biological properties
of these conjugates.
Methods. Firstly, PNVCL-COOH was synthesized by free
radical polymerization. Thereafter PNVCL-COOH was
conjugated with the natural biopolymers using EDC and
NHS. Phase transition temperatures were determined by
measuring the optical transmittance at 480 nm over the
temperature range of 20-50oC. The structures were
characterized by FTIR and DSC. The swelling kinetics of
the thermoresponsive biopolymers were determined. Cell
attachment and growth on the conjugates was evaluated
by MTT using rat bone marrow mesenchymal stem cell
cultures (BM-MSCs). Finally, the histocompatibility of the
thermoresponsive polymers was evaluated in the
subcutaneous rat model.
Results. Formation of the copolymers were confirmed by
FTIR. Both of the copolymers exhibited a temperaturedependent transition. The LCST values of the PNVCL-gcollagen and PNVCL-g-chitosan copolymers were found to
be 35oC and 31oC, respectively. Water uptake
experiments indicated that PNVCL-g-collagen was more
hydrophilic than PNVCL-g-chitosan. MTT findings
demonstrated that the copolymers supported the
attachment and growth of the cells, and were basically
not toxic to BM-MSC cultures. In-vivo studies were in line
with the in-vitro studies, thus the histological analysis did
not show any significant signs of inflammation and the
conjugates were quite well tolerated by the subjects.
Conclusions. The water-soluble and non-toxic
thermoresponsive polymer, PNVCL-g-collagen had a LCST
value (35oC) closer to the physiological temperature. This
feature may have an advantage in manipulating
mammalian cell cultures that are more sensitive to
temperature fluctuations
Keywords.
Thermoresponsive
polymers,
poly(Nvinylcaprolactam)-g-collagen, cell sheet engineering,
polymer brushes.
(15.P10) OSTEOBLASTIC DIFFERENTIATION OF BONE
MARROW STROMAL CELLS WITH GFOGER PEPTIDE ON
FIBRIN SCAFFOLDS
Herrera M (1), Jarquin K (1), Hernandez B (1), Medrano J
(1), Piñon G (1), Alvarez J (1), Acevedo S (1), Canchola E
(1), Sampedro E (1), Castell A (1)
1. Medicine School, UNAM
Implants of scaffolds of several biomaterials like calcium
phosphate slurries have been broadly used on diverse
therapeutic protocols for severe bone tissue damage,
acting not only as a mechanical support but promoting a
short time cell migration process of resident cells form
neighborhood healthy bone tissue into the implant, by
other side it has been demonstrated that the use of
collagen mimetic peptides whit sequences as GFOGER
which act as integrins receptors enhance migration and
proliferation. However osteoblastic differentiation do not
occur over this kind of scaffolds. In this study we use an
hydrogel of fibrin in combination with GFOGER peptide to
induce osteoblastic differentiation on bone marrow
stromal cells (BMSC).
Mice bone marrow were collected from tibia and femur
of five individuals by experiment, cell suspension was pre
cleared by 100 um filtration, seeded on DMEM/SFB 10%
and cultured overnight permitting the adhesion of BMSC
to the plate, myeloid and blood cells were discarded and
adherent cells harvested and transferred into a fibrin
solution supplemented with GFOGER peptide, finally
fibrin solution was solidified with calcium chloride and
the resultant hydrogel was cultured during 24 days on
DMEM/dexamethasone/ascorbic acid. After culture
period hydrogels were processed for transmission and
scanning electron microscopy and paraffin histology.
After 14 days BMSC seeded on fibrin hydrogel with
GFOGER shown a rounded morphology and their extra
cellular matrix resembles osteocyte lacunae, also
mineralization of extracellular matrix it was demonstrated
by the histology stain of von Kossa. Finally both 14 and 24
day of culture on fibrin-GFOGER, BMSC derived
osteoblastic-like
cell
were
positive
by
immunohistochemistry for osteopontin, and pro-collagen
type I.
Acknowledgegments. TH Raquel Guerrero Alquicira, M.
en C. Patricia Bizarro Nevares, CONACYT 50396-M,
DGAPA PAPIIT IN213510 e IN214109-3, Posgrado en
Ciencias Medicas Odontológicas.
Keywords. Fibrin, BMSC
(15.P11) α-HELICAL PEPTIDE HYDROGEL MATRICES FOR
3D CELL CULTURE AND TISSUE ENGINEERING
Mullen LM (1), Mehrban N (1), Banwell EF (2), Abelardo
ES (1), Birchall MA (3), Woolfson DN (1)
1. University of Bristol; 2. Tokyo Institute of Technology; 3.
University College London
Introduction. Tissue engineering (TE) promises to
regenerate a patient’s own tissue via the delivery of
scaffolds cells and biomolecules to the defect site.
Recently, self-assembling peptide hydrogels have shown
promise as TE scaffolds for such proposes. Potentially,
these systems offer reduced complexity, allow iterative
redesign and, ultimately permit recombinant production.
The work presented here investigates an α-helical dualpeptide system (hSAFs) for the 3D cell culture of a range
of different cell types, and evaluates the effect of
covalently tethered RGD motifs on cell adhesion and
migration [1].
Methods. Peptides were synthesised using standard
solid-phase 9-fluorenyl-methoxycarbonyl chemistry,
purified by reverse-phase HPLC, and confirmed by mass
spectrometry. CD Spectra were recorded between 190
and 260 nm using a Jasco J-810 circular dichroism
spectrometer. Fibres were imaged using negative stain
transmission electron microscopy. For gelation
experiments, 1 mM of each hSAF peptide was mixed on
ice for 5 min then incubated for 25 min at room
temperature, and overnight at 37 oC. PC12 cells were
cultured for 14 days.
Results. The hSAF system comprises two peptides with
coiled-coil repeats, which direct the assembly of a
heterodimeric interface and leave exposed surfaces to
promote weak hydrophobic interactions between fibrils.
These design features were tested and corroborated
using a combination of CD spectroscopy, electron
microscopy and rheology. The resulting hSAF hydrogels
support the growth and differentiation of PC12 cells.
Conclusion. We present a new self-assembling peptide
system, which forms hydrogels and has promise as
scaffolds for 3D cell culture and TE applications.
References. [1]. E. F. Banwell, E. S. Abelardo, D. J. Adams,
M. A. Birchall, A. Corrigan, A. M. Donald, M. Kirkland, L. C.
Serpell, M. F. Butler and D. N. Woolfson, Rational design
and application of responsive -helical peptide hydrogels,
Nature Materials 8, 596 - 600 (2009).
Keywords. Self-assembly, peptide, hydrogel
(15.P12) MODULATION OF INFLAMMATION TO
ENHANCE BONE REGENERATION BY DUAL DELIVERY OF
ANTI-IL-6 DRUG AND BONE MORPHOGENETIC PROTEIN2 WITH GELATIN HYDROGELS
Ratanavaraporn J (1), Tabata Y (1)
1. Institute for Frontier Medical Sciences, Kyoto University,
53 Kawara-cho Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
Introduction. Inflammation is a body response necessary
to start the natural process of tissue regeneration.
However, excessive inflammation responses sometimes
result in the suppression of regeneration process. In this
study, bone regeneration in the conditions of proinflammatory cytokines (e.g. IL-6 and TNF-α) suppression
was investigated to evaluate effect of inflammation
modulation on the process of tissue regeneration.
Methods. Triptolide of an anti IL-6 drug was encapsulated
in L-lactic acid-grafted gelatin micelles to allow it to
solubilize in water. Gelatin with the water-solubilized
anti-IL-6 drug in micelle was crosslinked by
glutaraldehyde to obtain gelatin hydrogels incorporating
anti-IL-6 drugs. After the subcutaneous implantation in
C57BL/6 mice, the number of inflammatory cells and the
expression of pro-inflammatory cytokine genes were
evaluated by flow cytometry and real-time polymerase
chain reaction (RT-PCR), respectively. The gelatin
hydrogels incorporating anti-IL-6 drug combined with
bone morphogenetic protein-2 (BMP-2) were implanted
into the ulna critical-sized defects of Wistar rats to
examine bone regeneration.
Results. The number of macrophages and neutrophils
infiltrated around the gelatin hydrogels incorporating
anti-IL-6 drug was significantly reduced comparing with
that of the drug-free gelatin hydrogels. The implantation
of gelatin hydrogels incorporating anti-IL-6 drug
significantly down-regulated the expression of IL-6 and
TNF-α genes, although the level was enhanced at higher
drug doses. The combinational application of anti-IL-6
drug and BMP-2 in gelatin hydrogels resulted in an
enhanced bone regeneration in the bone defect 4 weeks
post-operatively. However, when the dose of drug and
BMP-2 increased, no bone regeneration was observed.
Conclusions. An anti-IL-6 drug, Triptolide, showed a dosedependent effect on the modulation of inflammation and
bone regeneration. The gelatin hydrogels incorporating
an optimal combination of anti-IL-6 drug and BMP-2
efficiently enhanced bone regeneration.
Keywords. Inflammation, anti-IL-6, BMP-2, bone
regeneration
(15.P13) THE SIMULTANEOUS ENCAPSULATION OF
HUMAN ADIPOSE DERIVED STEM CELLS AND
TRANSFORMING
GROWTH
FACTOR
β1
IN
CARRAGEENAN HYDROGELS AS A NEW SYSTEM FOR
CARTILAGE TISSUE ENGINEERING
Rocha PM (1), Santo VE (1), Gomes ME (1), Reis RL (1),
Mano JF (1)
1. 3Bs Research Group - Biomaterials, Biodegradables and
Biomimetics, University of Minho
Introduction. The combination of hydrogels with stem
cells and growth factors (GFs) became a promising
approach to promote cartilage regeneration in the recent
years. Previous studies have shown the ability of human
adipose derived stem cells (hASCs) to differentiate
towards a chondrogenic lineage when entrapped in a
three-dimensional (3D) environment while Transforming
Growth Factor-β1 (TGF-β1) is one of the main GFs
influencing chondrogenic differentiation. Therefore, the
combination of both components encapsulated in a
temperature-responsive hydrogel is proposed in this
study as a new system for cartilage tissue engineering
(TE).
Methods. Carrageenan hydrogels (2 and 2.5% (w/v)) were
prepared by ionic gelation with potassium chloride. In a
preliminary study, ATDC5 cell line was encapsulated to
analyze the biomaterial cytotoxicity and the influence of
polymer concentration in cell viability and proliferation.
Furthermore, the encapsulation of hASCs was performed
together with the entrapment of TGF-β1 in the hydrogels
networks. The cells and TGF-β1 were quickly mixed with
the polymer solution around 40ºC and allowed to cool
down until gelation occurred. Afterwards, the constructs
were cultured in vitro up to two weeks in chondrogenic
and basal mediums. The gels encapsulating only hASCs
and cultured in chondrogenic medium were used as
controls. The constructs were then characterized for cell
viability,
proliferation,
histology
and
immunohistochemistry for cartilage specific markers.
Results. We demonstrated that κ-carrageenan is suitable
biomaterial for cell and GF encapsulation as cells remain
viable in culture for both ATDC5 and hASCs. The culture of
the constructs for 14 days in different conditions revealed
specific cartilage extracellular matrix molecules
expression by hASCs.
Conclusions. The incorporation of TGF-β1 within the
carrageenan-based hydrogel enhances the chondrogenic
differentiation of hASCs. These findings suggest a new
system for cartilage TE, which is even more promising for
future applications as an injectable system due to its
thermoresponsive behavior.
Acknowledgments. European NoE EXPERTISSUES (NMP3CT-2004-500283), European FP7 Project Find&Bind
(NMP4-SL-2009-229292),
FCT(PTDC/FIS/68517/2006,
PTDC/QUI/69263/2006, BD/39486/2007), Hosp. da
Prelada.
Keywords. Adipose derived stem cells, Transforming
Growth Factor, hydrogel, cartilage tissue engineering
(15.P14) ISOLATION OF HUMAN MESENCHYMAL
STROMAL CELLS ON HYDROGEL SCAFFOLDS WITH
VARYING STIFFNESS AND LIGAND FUNCTIONALISATION
Schneider K (1), Pompe T (1), Freudenberg U (1), Müller K
(2), Bornhäuser M (2), Werner C (1,2)
1. Leibniz Institute of Polymer Research Dresden, Max
Bergmann Center of Biomaterials, Dresden; 2. Technische
Universität Dresden, Center for Regenerative Therapies
Bone marrow-derived multipotent mesenchymal stromal
cells (MSCs) are able to self-renew and differentiate into a
variety of cell types of mesodermal as well as into nonmesodermal origin making them attractive for studies in
regenerative medicine. Current isolation protocols
generate rather heterogeneous populations using percoll
density gradient centrifugation of bone marrow aspirates
and subsequent adhesion and expansion on tissue culture
plastic. Since recent studies assigned material properties
such as elasticity and ligand presentation an important
role in MSC differentiation in vitro, we aim at establishing
a new expansion strategy to identify and harvest distinct
subpopulations of MSCs.
Layers of biohybrid heparin and multi-armed
poly(ethylene glycol) hydrogels are utilized to adjust
stiffness and adhesion ligand presentation in defined
ways. The hydrogel layers were produced in thicknesses
of about 50 µm and with degrees of crosslinking resulting
in elastic moduli between 2 and 42 kPa. Adhesion
peptides consisting of amino acid sequences found in the
extracellular matrix components fibronectin, collagen,
and laminin were covalently conjugated to the heparin
units of the gels. The results demonstrate that the
compared variants of the gel materials can effectively
modulate the adhesion, proliferation, and differentiation
of MSCs. Based on that, current studies concern the
application of the hydrogel surfaces to isolate distinct
MSC subpopulations out of primary density gradients of
bone marrow aspirates. The MSCs expanded on hydrogel
surfaces are thoroughly characterized using flow
cytometry and clonal as well as differentiation assays.
Taken together, a set of gradually adjusted biohybrid
hydrogel materials is used as layered cell culture carrier
system to isolate, maintain, and expand human MSCs
offering valuable options for the more targeted
application of the marrow-derived cells in regenerative
therapies.
Keywords. Biohybrid starPEG/Heparin hydrogel, MSC
(15.P15) ESTABLISHMENT OF AN IN VITRO MODEL WITH
HUMAN
MESENCHYMAL
STEM
CELLS
AND
MICROVASCULAR ENDOTHELIAL CELLS FOR MENISCAL
REPAIR
Weyhmüller J (1), Rücker C (1), Steinert A (2), Rudert M
(2), Walles H (1), Heymer A (1)
1. Chair Tissue Engineering and Regenerative Medicine,
University Clinic of Würzburg, Germany; 2. Orthopedic
Center for Musculoskeletal Research, Division of
Gentherapy, University of Würzburg, Germany
Introduction. To date, tissue engineering constructs for
meniscus regeneration failed, due to their limited
integration capacities. A vascularised cell-based meniscus
graft should overcome these limitations. Therefore,
human mesenchymal stem cells (hMSCs) were embedded
in three-dimensional (3D) collagen matrices in vitro to
evaluate the optimal scaffold with homogenous cell
distribution. Additionally, a co-culture-system of hMSCs
and human microvascular endothelial cells (hmvECs) was
established to predict the interactions between both cell
types.
Methods. HMSCs isolated from bone marrow were
seeded with various cell concentrations onto 5 different
collagen matrices, fibrous as well as hydrogels. With
histological stainings (HE and live-dead-staining) the cell
distribution and viability in the matrix were visualized. To
characterize hMSCs in the construct immunhistological
analysis was performed. To build-up the co-culturesystem, first investigations were made by seeding hMSCs
and hmvECs in hanging inserts. The cell proliferation was
compared to the normal tissue culture surface using a
WST-1 assay. Further both cell types were cultured in
different mixtures of the cell-type-specific media to
evaluate the optimal medium composition.
Results. A homogeneous cell distribution was attained by
seeding 500,000 cells/ml collagen-I-hydrogel as well as on
50 mm2 collagen-I-electrospun matrix. The cells were
viable, even inside the matrix. The functionality of hMSCs
was demonstrated by the synthesis of collagen-I. All other
tested matrices showed an hMSC monolayer on the outer
edge of the construct but no cells inside the scaffold. The
co-culture-system was established with hMSCs on the
insert membrane and hmvECs on the tissue culture
surface because of the limited proliferation of hmvECs on
the insert membrane. As culture medium a mixture 10:1
of endothelial to hMSC medium showed only minor
impact on cellular behaviour for both cell types.
Conclusions. Further investigations have to show whether
a co-culture of hMSCs and hmvECs in 3D constructs has
an influence on the differentiation of the cells.
Keywords. Vascularised meniscus tissue, in vitro 3D
model, stem cells, scaffold
(15.P16) ENHANCED SKIN WOUND HEALING BY A
SUSTAINED RELEASE OF GROWTH FACTORS IN
PLATELET-RICH PLASMA
Yang HS (1), Shin J (2), Bhang SH (2), Shin JY (2), Kim BS (2)
1. Department of Bioengineering, Hanyang University, 17
Hangdang-Dong, Sungdong-Gu, Seoul 133-791, Republic
of Korea; 2. School of Chemical and Biological
Engineering, Seoul National University, San 56-1, Silimdong, Gwanak-Gu, Seoul 151-744, Republic of Korea
Introduction. Platelet-rich plasma (PRP) contains growth
factors that can promote tissue regeneration. Previously,
we have shown that heparin-conjugated fibrin (HCF) can
exert a sustained release of growth factors that have
affinity to heparin. Here, we hypothesized that treatment
of skin wound with a mixture of PRP and HCF would exert
a sustained release of several growth factors contained in
the PRP and promote the skin wound healing.
Methods. PRP was prepared by centrifuging whole blood
at 2,400 rpm for 10 min and subsequently 3,500 rpm for
15 min. Full-thickness (2.0 x 2.0 cm) wounds were created
on the dorsum of athymic mice. HCF mixed with PRP and
thrombin was applied at the wound sites. No treatment,
application of PRP with thrombin, PRP with fibrinogen
and thrombin served as controls. Skin regeneration was
evaluated by histological and immunohistochemical
analyses.
Results. The release of fibroblast growth factor 2 (FGF2),
platelet-derived growth factor-BB (PDGF-BB), and
vascular endothelial growth factor (VEGF) contained in
PRP from HCF was sustained for a longer period than that
from either PRP only, C-PRP, or a mixture of F-PRP in
vitro. At 12 days after injury, PRP with HCF group showed
complete epithelialization of the wound compared to the
other groups. The macroscopic wound sizes of PRP with
HCF group were statistically smaller than the other groups
at 12 days. The HCF with PRP groups showed excellent
epithelial maturation.
Conclusion. The enhanced skin regeneration in HCF-PRP
group may be at least partially due to enhanced
angiogenesis in the wound beds. This method could be
useful for skin wound treatment.
This work was supported by the Korea Health 21 R&D
project, Ministry of Health and Welfare (A100443).
Keywords. Growth factors; Heparin-conjugated fibrin;
Platelet-rich plasma; Skin wound healing
(15.P17) LOCAL TRANSFORMING GROWTH FACTOR-BETA
DELIVERY IN FIBRIN HYDROGEL: RELEASE KINETICS AND
EFFECTS ON HUMAN MESENCHYMAL STEM CELL
CHONDROGENESIS
Diederichs S (1), Baral K (1), Tanner M (1), Richter W (1)
1. Research Center for Experimental Orthopaedics,
University of Heidelberg
Introduction. Structural extracellular matrix molecules
gain increasing attention as scaffolds for cartilage tissue
engineering due to their natural role as a growth factor
repository. We recently observed that a collagen type I/III
matrix (Col-I/III), human recombinant TGF-β protein, and
fibrin glue (FG) combined to a biphasic construct provided
sufficient long-term TGF-β support to drive in vitro
chondrogenesis of human mesenchymal stem cells (MSC)
for 6 weeks. Here we ask whether FG and Col-I/III can
both retain TGF-β, describe the influence of cells on TGFβ release and compare the quality of chondrogenic
differentiation of human MSCs between soluble versus
local TGF-β supply.
Methods. Release of growth factor from mono- and
biphasic scaffolds augmented with increasing amounts of
TGF-β was analysed over 7 days and chondrogenesis was
assessed over 42 days.
Results. Low TGF-β release rates from Col-I/III as opposed
to higher release from FG indicated that both molecules
retained TGF-β, with Col-I/III being the superior storage
component. Embedding of cells significantly reduced the
cumulative TGF-β release but fibrin-entrapped TGF-β
remained bioactive and supported MSC chondrogenesis
similar to standard scaffold-free MSC pellets supplied
with soluble TGF-β. FG plus soluble TGF-β allowed
significantly more proteoglycan and collagen type II
deposition per construct than FG plus local TGF-β and
pellet controls. However, less collagen type X relative to
collagen type II and no MMP-13 was induced at local TGFβ supply suggesting a reduced hypertrophy during
chondrogenesis.
Conclusions. Local growth factor application, thus, opens
an interesting new perspective to modulate
differentiation routes between more stable as opposed to
transient cartilage.
Keywords.
human
mesenchymal
stem
cells,
chondrogenesis, fibrin hydrogel, transforming growth
factor beta, collagen type X
(15.P19) NON-COVALENTLY CROSS-LINKED HYDROGELS
FOR APPLICATIONS IN REGENERATIVE MEDICINE
Pashuck ET (1), Clarke DE (1), Gentilini C (1), Stevens MM
(1)
Introduction. Numerous approaches have been used to
create three-dimensional scaffolds for tissue engineering,
including electrospun polymers, polymer hydrogels and
self-assembled peptides. While each of these approaches
is promising, they can be hindered by things such as
initiator toxicity for polymeric hydrogels and degradation
of mechanical properties at low strains for self-assembled
materials. Designing an injectable material that combines
the strain resistance of polymeric materials but can be
gelled without initiators would be an important advance
in the field of biomaterials. We have designed a novel
system that consists the biopolymer poly (γglutamic acid)
(γ-PGA) functionalized with self-assembling peptide
groups that are designed to act as non-covalent crosslinks, as depicted in Figure 1a.
Methods. The β-sheet peptides of interest had N-terminal
cysteines that were covalently coupled to poly (γglutamic
acid) (γ-PGA) functionalized with maleimides. The
polymer-peptide composite was dissolved in water at a
physiological pH. A549 lung carcinoma cells were seeded
into the peptide-γ-PGA gels and covered with media. Cell
viability was assessed using a LIVE/DEAD stain containing
calcein AM and ethidium bromide, respectively.
Results. We have successfully modified γ-PGA with a selfassembling peptide using thiol-maleimide chemistry.
These peptide-polymer composites form a hydrogel with
just a few percent of the carboxylic acids functionalized
with the β-sheet peptide. The hydrogel was capable of
being stretched and manipulated with tweezers, as
shown in Figure 1b. Figure 1c shows that cells
encapsulated in the hydrogel showed good viability in the
scaffold after 18 hours. Cell differentiation assays with
mesenchymal stem cells are ongoing.
Conclusions. Non-covalent cross-linking of biopolymers is
a promising method for engineering enzymatically
biodegradable hydrogels for applications in regenerative
medicine. This allows for minimally invasive injectable
therapies and functionalization of the scaffold with
orthogonal chemistries, such as click-chemistry, allows for
multiple bioactive groups to be incorporated on a single
scaffold.
Keywords. Hydrogel, self-assembly, polymer, stem cells
(15.P20) SKELETAL IN SITU TRANSDIFFERENTIATION OF
ASCS IN A 3D CULTURE SYSTEM
Nieto-Aguilar R (1), Serrato D (1), Fernández-Valadés R,
Martín-Piedra MA (1), Carriel V (1), Garzón I (1), Campos
A (1)
1. Tissue Engineering Group, Department of Histology,
University of Granada, Granada, Spain; 2. Division of
Pediatric Surgery, University Hospital Virgen de las Nieves,
Granada, Spain
Introduction. Generation of skeletal tissues by tissue
engineering should use a single and accessible cell source
and an adequate tree-dimensional scaffold biomaterial. In
this work, we have developed a novel model of 3D bone
and cartilage tissue substitutes by means of
transdifferentiation of ASCs using fibrin-agarose
hydrogels.
Methods. ASCs primary cultures were obtained from
human biopsies of subcutaneous adipose tissues. After
ASCs cultures reached subconfluence, the cells were
seeded in a three-dimensional scaffold which consisted of
human fibrin and agarose type VII using DMEM as basal
culture medium. To generate bone and cartilage-like
tissues, constructs were induced to the osteogenic and
chondrogenic lineages for 21 days. Then, samples were
obtained after 24 hours; 7, 14 and 21 days and processed
for histological and immunofluorescence analyses to
verify the transdifferentiation process using alizarin red S
and alcian blue stains for histochemestry and alkaline
phosphatase and collagen type II antibodies for
immunofluorescence correspondingly.
Results. After 14 days of induction bioengineered tissues
induced to both, the osteogenic and the chondrogenic
lineages showed an incipient osteogenic and
chondrogenic differentiation as determined by alizarin
red S and Alcian blue staining. After 21 days of induction
the synthesis of both the calcic and mucopolysaccharides
materials increased. Immunofluorescence revealed a high
alkaline phosphatase activity after 14 and 21 days of
osteoinduction whereas collagen type II showed an
incipient signal after 7 days and increased after 14 and 21
days.
Conclusions. Fibrin-agarose 3D culture model could
support the efficient transdifferentiation capability of
ASCs, suggesting that the generation of threedimensional human tissue substitutes using fibrin-agarose
scaffolds is a feasible technique in the laboratory and
could be used in regenerative medicine.
Acknowledgments. This work was supported by CTS-115
Tissue Engineering Group.
Keywords. Engineered skeletal, tridimensional scaffold,
pluripotent
16. ENGINEERING A FUNCTIONAL
TENDON
Chair: Dimitrios I Zeugolis
Co-chair: Oded Shoseyov
Keynote speaker: Wei Liu
Organizers: Dimitrios I. Zeugolis, Oded Shoseyov
Synopsis: Advances in medical care have greatly
improved survival rate and life expectancy following
trauma or degenerative conditions of the musculoskeletal
system, leading to an ever-increasing need for functional
tissue substitutes to improve quality of life. Tendon and
ligament injuries constitute the most common
musculoskeletal disorders that clinicians address daily.
Indeed, from over 33 million musculoskeletal injuries per
year in United States alone; almost 50% of them are
tendon and ligament related with approximately 95,000
new cases per year. As tendons have limited regeneration
capacity, suitable substitutes are required for
regeneration and functional recovery. Surgical repairs are
still suboptimal due to fibrous adhesions or failure arising
from the mechanical demands placed on imperfect
integrative healing at tendon-tendon or tendon-bone
interfaces. Therefore, to develop strategies for functional
tendon regeneration is of paramount importance.
However, in order to be able to imitate nature, we need
to understand how the tissue forms and behaves in vivo
under normal and pathophysiological conditions. Upon
this knowledge, we can develop strategies that will
encourage scaffold interaction with extracellular matrix
components, growth factors, cells and cell surface
receptors. We will also be able to identify bioactive and
therapeutic molecules that should be incorporated into
the 3D construct that will positively interact with the host
and promote functional regeneration. This symposium
will discuss current tissue engineering strategies that
improve tendon regeneration and functional recovery by
using recent advancements in material optimisation and
scaffold functionalisation through incorporation of
biophysical cues and biochemical and biological signals.
(16.KP) DERMAL FIBROBLAST BASED TENDON
ENGINEERING: FROM BASIC RESEARCH TO PRE-CLINICAL
TRIALS
Liu W (1), Cao Y (1)
1. Shanghai Jiao Tong University School of Medicine.
Shanghai, China
One of the major challenges in tendon engineering is the
selection of proper cell source. In our group, we explored
the possibility of using dermal fibroblasts as the cell
source for tendon engineering. At the cellular level, the
fibroblasts were forced into an elongated morphology
and then subjected to a unilateral mechanical stretch.
The results demonstrated that dermal fibroblasts could
actually be transdifferentiated into tenocytes by
expressing tenogenic markers such as tenomodulin,
tenacin, deocrine, collagen VI. Interestingly, the tenogenic
transdifferentiation became most prominent only when
the mechanical force was applied parallel to the long axis
of the elongated cells. At the tissue level, it was found
that human fibroblasts could form better tissue structure
when they were seeded on a parallel aligned polymer
fiber scaffold than on randomly arranged fibers.
Furthermore, during in vitro culture, the mechanical
loading resulted in better engineered tendon tissue than
non-loaded tissues including stronger mechanical
strength, better tissue structure, and thicker collagen
fibrils. Quantitative analysis revealed no difference in
above mentioned characters between human dermal
fibroblast and tenocyte engineered tendons, indicating
the importance of mechanical loading in transforming
fibroblasts to tenocytes and tendon tissue formation. To
further translate the finding to pre-clinical study, a
complex scaffold with reinforced mechanical strength
was fabricated and used for in vitro tendon engineering
along with dermal fibroblasts. The results showed that
the tensile strength of the in vitro engineered tendon
could reach about 50 Newtons. Then the tendon graft
was implanted in vivo for repairing the flexor tendon
defect in a monkey model. After 6 months, the repair
flexor tendon could regain its major function.
Keywords. Dermal fibroblast, tendon engineering, in
vitro, transdifferentiation
(16.O1) TENOMODULIN PROMOTES THE TENOGENIC
DIFFERENTIATION OF MESENCHYMAL STEM CELLS
Jiang Y (1), Zhou G (1), Zhang W (1), Cao Y (1), Liu W (1)
1. Shanghai Key Laboratory of Tissue Engineering
Research, Shanghai, China
Aim. Tendon tissue engineering provides a promising
approach for tendon defects. While the mechanism of
development and maturation for engineered tendon
remains unknown, due to lack of specific tendon related
markers.
Recently
tenomodulin
(TNMD)
was
demonstrated to be a relative specific tendon marker.
However, the effect of TNMD on the tenocyte or
mesenchymal stem cells (MSCs) and its’ possible
application in tendon tissue engineering are unexplored.
This study employed gene transfection to investigate the
effects of TNMD on the tendon precursor cells (TT-D6)
and MSCs (C3H10T1/2).
Method. The in vitro cultured TT-D6 cells and C3H10T1/2
cells were stable transfected with pCAGGS-TNMD using
the Fugene HD. After confirmation of successful
transfection, the morphology, the proliferation and the
expression of some tendon related genes were analyzed.
At the same time, the multilineage differentiation of
C3H10T1/2 cells after transfection was also tested.
Results. Successful overexpression of TNMD was
confirmed by RT-PCR, quantitative PCR and
immunofluorescent staining. No obvious differences in
morphology were seen in both normal TT-D6 cells and
C3H10T1/2 cells after transfection. While faster
proliferative ability was shown in both cells. For TT-D6
cells transfected with pCAGGS-TNMD, the expression of
collagen1, collagen3, collagen6, biglycan and decorin
were up-regulated significantly. The expression of
scleraxis, COL1, COL3, and decorin were increased
dramatically, while the expression of COL6 and biglycan
were not influenced obviously in C3H10T1/2 cells.
Differentiation test indicated that TNMD could inhibit the
adipogenic
differentiation,
the
chondrogenic
differentiation and AKP activity of C3H10T1/2 cells.
Conclusions. These findings demonstrate that TNMD can
enhance the expression of the extracellular matrix of the
tendon precursor cells and promote the the tenogenic
differentiation of mesenchymal stem cells, which may
indicate the possible application of TNMD in tendon
tissue engineering and tendon repair.
Keywords. Tenomodulin tendon transfection
(16.O2) CURRENT CLINICAL OPINION OF ANTERIOR
CRUCIATE LIGAMENT TISSUE ENGINEERING
Rathbone SR (1), Maffulli N (1), Cartmell SH (2)
1. University of Manchester; 2. Queen Mary University of
London, Barts and The London School of Medicine and
Dentistry, London
Introduction. Donor site morbidity, poor graft site
integration (causing slippage) and incorrect mechanical
performance are all common problems with grafts
currently used for repairing the anterior cruciate ligament
(ACL). A tissue engineered (TE) ligament has potential to
overcome these problems. We have obtained input from
clinicans who currently treat these injuries to deal with
any potential design short-comings associated with TE
ACLs before they arise.
Methods. An online questionnaire was created relating to
ACL tissue engineering. The questionnaire was peer
reviewed and approved by local research ethics
committee (project number 09/H1204/64, approved
15/10/09). Between July and October 2010, three
hundred orthopaedic surgeons specialising in ligament
and tendon repair in the UK were contacted by email and
invited to participate. From this e-mailed input request,
eighty surgeons responded.
Results. 86% of surgeons would consider using a TE ACL if
it were an option (provided it showed biological &
mechanical success) if it significantly improved the
patient satisfaction (63%) and shortened surgical time
(62%), and would be prepared to wait 4-30 weeks for it to
be created. 42% were either concerned or very concerned
(25%) about its successful integration into the bone. 76%
of surgeons felt that using a TE ACL would be more
appropriate than a patellar tendon, hamstring or
quadriceps autograft if it could be engineered to be an
exact match to the native tissue. 62% thought using a TE
ligament would take less surgical time & felt surgical time
needs to be reduced by 10-30 minutes to be considered
to be a significant improvement.
Conclusions. Overall it appears that most surgeons would
be prepared to use a TE ligament. Future research needs
to concentrate on integration of the TE ACL into the
patients' bone. This information confirms a demand for
tissue engineered ACL’s and highlights important areas
for improvement.
Keywords. Survey, anterior cruciate ligament, tissue
engineered construct, implantation
(16.O3) IMPLANTATION OF ELECTROSPUN POLY(εCAPROLACTONE ) 3D SCAFFOLDS IN ACHILLES TENDONS
Bosworth LA (1), Alam N (2), Downes S (1)
1. School of Materials, The University of Manchester, UK;
2. Blond McIndoe Laboratories, School of Medicine, The
University of Manchester, UK
Introduction. Tendons are susceptible to wear and tear
and even spontaneous rupture. There remains an unmet
clinical need for the development of a medical device
capable of regenerating damaged tendons. We have
fabricated an electrospun, synthetic, biodegradable
scaffold with proven biocompatibility and appropriate
interface when grafted in vivo.
Methods. Electrospun fibres were fabricated using poly(εcaprolactone) (Mn 80,000) dissolved in Acetone
(concentration 10%w/v) and parameters: voltage - 20kV,
flow rate - 0.05ml/min, distance to collector – 15cm. 2D
fibre mats were manipulated into 3D fibrous scaffolds.
Scaffold biocompatibility with tenocytes was assessed in
vitro over 14 days by cell proliferation and topographical
cues. In a pilot animal study, partial removal of the
Achilles tendons’ of mice was performed to create a
critical defect, which was grafted with a single
electrospun 3D scaffold. Mice were monitored for 21 days
and assessment of the scaffold-tissue interface was
determined by variable-pressure Scanning Electron
Microscopy.
Results. Tenocytes adhered and proliferated along the
longitudinal axis of scaffold fibres, demonstrating that
fibres provided adequate topographical cues to guide cell
orientation. This was further confirmed by comparison to
cells cultured on randomly orientated electrospun fibres.
In all mice receiving scaffold grafts, normal ambulation
was observed within 48hrs post-operatively. Initially there
was good interaction at the tissue-scaffold interface
(Figure), and by day 21 post-implantation the scaffold had
been fully integrated into the tendon, suggesting new
tissue formation. All mice survived the time period
investigated.
Conclusion. This study has demonstrated the
biocompatibility and successful implantation of 3D
electrospun scaffolds into the Achilles tendons’ of mice.
On-going work performing long-term in vivo assessment
and histomorphometric analysis will advance this
technology towards our ultimate goal of clinical tendon
regeneration.
Acknowledgements. The Authors wish to acknowledge
RegeNer8 and the UMIP Premier Fund for funding this
research.
Keywords. Electrospinning, polycaprolactone, tendon,
nanofibres
(16.O4) GEL SPINNING OF ALIGNED HUMAN
RECOMBINANT COLLAGEN FIBERS BY INJECTION OF
NEMATIC LIQUID CRYSTALLINE DOPE
Yaari A (1), Shoseyov O (2)
1. Collplant Ltd. Ness-Ziona, Israel; 2. The Robert H. Smith
Faculty of Agricultural, Food and Environment Quality
Sciences. The Hebrew university of Jerusalem. Rehovot,
Israel
Collagen is the most abundant structural protein in
mammals. It is a key component in load bearing tissues
(Ligaments and bones), giving them tensile strength and
resilience. The unique mechanical properties of these
tissues are dependent on the highly ordered and
hierarchical organization of the collagen fibers.
There are significant advantages for the use of collagen
based scaffolds for tissue repair because of its
biocompatibility and biodegradability, but so far there
was only a limited success in creating structures that
would have the required strength. This is partly attributed
to a lesser degree of order achieved so far in artificial
collagen fibers.
Above a threshold concentration collagen solutions
become liquid crystalline, and that property plays a key
role in the formation of certain tissue structures, such as
the dogfish egg capsule. In this work, we utilize collagens
unique liquid crystalline and self assembly properties to
create ordered scaffolds for tissue replacement.
Liquid crystalline human recombinant collagen dope, in
acidic pH, was injected through a 30 ga. needle that
exerts strong shear force on it. A nematic order was
induced in the outer layer of collagen rod-like molecules,
which then spreads through the injected material as
determined by polarized light microscopy and crosssection SEM analysis. The fibers were extruded into a
coagulation bath that induces fibrillogenesis, a sol-gel
transition. A neatly ordered array of aligned collagen
fibrils is with the characteristic D-banding obtained, as
determined by high resolution SEM and AFM (figure 1).
Crosslinking can be performed by a plurality of methods,
to improve the fibers mechanical properties or reduce
swelling and biodegradation.
The fibers thus obtained display good mechanical
performance, including a UTS similar to natural rat tail
tendon (350 MPa), and may find applications in
regenerative medicine in general and tendon repair in
particular.
Keywords. Recombinant Human Collagen, Tendon Repair,
Liquid Crystall, Gel Spinning
(16.O5) EVALUATION OF CELLULAR FUNCTIONS AT THE
NANO-BIO-INTERFACE
English A (1), Rooney N (2), Pandit A (1), Zeugolis D (1)
National University of Ireland Galway, Ireland; 2. Proxy
Biomedical
Introduction: Cell-substrate interactions at the nano-bio
interface are becoming increasingly important in our
understanding of a range of physiological processes.
Indeed, nano-textured biomaterials have been shown to
favourably promote cell attachment, migration and
differentiation, since they closely imitate the in vivo
niche. However, to facilitate clinical translation of such
technologies, it is important to comprehend the influence
of nano-topography on the cellular and molecular level,
and to use this knowledge to design the next generation
of nano-biomaterials. Herein, the influence of nanotopography, induced by different scaffold conformations,
on cellular function was studied.
Methods: PLGA nano-textured scaffolds were produced
using solvent casting, electro-spinning, laser and nanoimprinting lithography. Human primary (WI38 lung
fibroblast) and immortalised (SAOS2 Osteosarcoma) cells
were seeded on the fabricated scaffolds for 2 to 14 days.
Subsequently, the influence of surface topography on cell
behaviour (e.g. morphology, attachment, alignment,
migration, phenotype maintenance) was evaluated.
Results: Scanning electron micrographs show the various
conformations of scaffolds used in this study (Figure-1.1).
Rhodamine-phalloidin and DAPI staining (Figure-1.2)
demonstrate that only aligned electro-spun nano-fibrous
mats (Figure-1.2.c) facilitated cell attachment and
alignment in the direction of the nano-fibrous substrate.
The metabolic activity of SAOS2 was significantly lower
(p<0.05) on electro-spun mats than on tissue culture
plastic at day 10 and 14 (Figure-1.3). Similarly to aligned
electro-spun nano-fibrous mats, nano-imprinted scaffolds
facilitated cell attachment and alignment and exhibited a
decrease in metabolic activity of WI38 fibroblasts at day 5
and 7 (data not shown).
Conclusions: Aligned electro-spun mats and nanoimprinted films provide a conductive environment for cell
attachment and orientation, whilst decreasing metabolic
activity. Studies are underway to understand the effect of
nano-topography at the molecular level to decipher the
effect on gene expression.
Acknowledgments. This work was supported by
Enterprise Ireland, CCAN (Project No. CCIRP-2007-CCAN0509) and by the Irish Government under the NDP 20072013.
Keywords. Nanotopography, Electro-spinning, Nanoimprinting; Cell Behaviour; Gene Expression
Figure 1.1 shows scanning electron micrographs of (a)
solvent casted films; (b) non-aligned electro-spun nanofibrous mats; and (c) aligned electro-spun nano-fibrous
mats. (d), (e) and (f) represent laser lithography treated
(a), (b) and (c) samples respectively. Figure 1.2:
Immunocytochemistry images illustrating the cellular
attachment and orientation on the scaffolds that were
viewed in figure 1.1. Figure 1.3: Cell metabolic activity
assay for osteosarcoma cells (SAOS2) on electrospun
mats. Control: Tissue Culture Plastic.
(16.P1)
POLY(3-HYDROXYBUTYRATECO-3HYDROXYHEXANOATE) (PHBHHX) SCAFFOLDS FOR
TENDON REPAIR IN THE RAT MODEL
Lomas AJ (1), Webb WR (1), Zeng G (2), Forsyth NR (1), El
Haj AJ (1), Chen GQ (2)
1. Keele University, UK; 2. Tsinghua University China
Introduction.
Poly(3-hydroxybutyrate-co-3hydroxyhexanoate) (PHBHHx) was investigated for
possible application in repairing damaged tendon, with a
range of in vitro and in vivo experiments utilised to design
and test suitable scaffold designs.
Methods. Scaffolds consisting of fibre reinforced porous
tubes were prepared using particle leaching and an
extrusion method. Mechanical testing demonstrated that
PHBHHx scaffolds could be produced that had
comparable mechanical properties to natural Rat Achilles
tendon. Sprague–Dawley (SD) rats were split into 3
experimental groups; no construct/control, PHBHHx
scaffold, and PHBHHx scaffold/collagen hybrids. The in
vivo functionality of scaffolds was explored in a surgically
induced Achilles tendon defect model, with polymer
breakdown products and C-Reactive protein blood plasma
concentrations measured throughout the experiment.
Mechanical testing and histological analysis was
performed after animal sacrifice at day 40.
Results. Mechanical tests demonstrated that the PHBHHx
scaffolds had comparable mechanical properties to
natural tendon, with maximal loads of 23.73 ± 1.08N,
compared to 17.35 ± 1.76N in undamaged rat Achilles
tendon. Restoration of movement and mechanical
loading was restored in scaffold-recipient rats at an
earlier time than those without scaffold, with almost
complete motion returning 10 days post surgery
compared to 20 days in the control. In vitro mechanical
testing of day 40 tendons demonstrated that the repair
induced in the scaffold/collagen model was comparable
to undamaged tendon (18.02 ± 7.45N vs. 17.35 ± 1.78N)
and integration was observed. Histological analysis of the
damaged area found evidence for tenocyte invasion
coupled with tissue remodelling. No significant secondary
immune response to PHBHHx was observed over time
with blood C-reactive protein levels remaining at control
cell levels throughout. In addition, measurement of Bhydroxybutyrate (a degradation product of PHBHHx)
blood concentration demonstrated no correlation to
immune response.
Conclusions. PHBHHx collagen hybrids have been
successfully used as a material for tendon tissue
engineering in vivo.
Keywords.
Polyhydroxyalkanoates,
Tendon,
Immunological Response, Tissue Engineering
17. ENGINEERING BIOMIMETIC
SCAFFOLDS FOR IN VITRO STUDIES
AND REGENERATIVE THERAPIES
Chair: Helena S. Azevedo
Co-chair: Alvaro Mata
Keynote speaker: Matthias Lutolf
Organizer: Helena S. Azevedo, Alvaro Mata
Synopsis: Recent advances in biomaterials research have
enabled engineering of scaffolds that reproduce the
biological, physical and biochemical complexity of the
natural extracellular matrix (ECM) environment. Within
regenerative medicine, the role of the scaffold is essential
and continues to increase primarily because of our
growing ability to create materials that can mimic the
natural ECM and elicit specific biological responses. The
opportunity to create scenarios that are bioactive and
biomimetic in the laboratory offers very attractive
opportunities for many in vitro applications. For example,
scaffolds that facilitate the execution of systematic
studies to elucidate the molecular mechanisms
underlying physiological and pathological processes,
deconstruction of complex biological processes or
extracellular environments, and analysis of specific signals
or mechanisms that affect cell-cell or cell-ECM
interactions. Biomimetic structures that better recreate in
vitro the complex natural in vivo environment have
tremendous implications in the design of novel therapies,
drugs, tissue engineering or regenerative medicine
scaffolds, or biomaterials while decreasing the need for in
vivo testing.
State-of-the-art scaffolds for such in vitro applications
vary widely from highly porous biodegradable supports,
bioactive substrates or smart drug-eluding materials, to
self-assembling hydrogels, precise microfabricated
structures, or lab-on-hip devices. Therefore, this
symposium will focus on work that features
bioengineering approaches to develop novel scaffolds
(defined as substrates, structures, or matrices), designed
to recreate the hierarchical complexity of tissues, to study
cell behaviour in vitro, learn fundamental processes for
future regenerative therapies, or aid the design or
effectiveness of regenerative therapies such as expanding
cell populations for cell therapies or facilitating new drug
discoveries. The symposium will cover the following
topics:
- Scaffolds that facilitate the deconstruction of biological
processes or cellular microenvironments to study cell
behaviors and learn basic embryogenesis, homeostasis, or
regeneration mechanisms.
- Scaffolds that facilitate the controlled growth of cell
populations for cell therapies.
- Scaffolds that facilitate the recreation of environments
for drug discovery.
(17.KP1) DESIGNING SMART
INSTRUCT STEM CELL FATE
Lutolf MP (1)
BIOMATERIALS
TO
1. Laboratory of Stem Cell Bioengineering, EPF Lausanne,
Switzerland
Proper tissue maintenance and regeneration relies on
intricate spatial and temporal control of biochemical and
biophysical microenvironmental cues, instructing stem
cells to acquire particular fates, for example remaining
quiescent or undergoing self-renewal divisions. Despite
rapid progress in the identification of relevant niche
proteins and signaling pathways using powerful in vivo
models, to date, many adult stem cell populations cannot
be efficiently cultured in vitro without rapidly
differentiating. To address this challenge, we and others
have been developing biomaterial-based approaches to
display and deliver stem cell regulatory signals in a
precise and near-physiological fashion, serving as
powerful artificial microenvironments to study and
manipulate stem cell fate both in culture and in vivo. In
this talk I will highlight recent efforts in my laboratory to
develop microarrayed artificial niches based on a
combination
of
biomolecular
hydrogel
and
microfabrication/robotic technologies. These platforms
allow key biochemical characteristics of stem cell niches
to be mimicked and the physiological complexity
deconstructed into a smaller, experimentally amenable
number of distinct signaling interactions. The systematic
deconstruction of a stem cell niche may serve as a
broadly applicable paradigm for defining and
reconstructing artificial niches to accelerate the transition
of stem cell biology to the clinic.
(17.KP2) SOFT NANOSTRUCTURE BIOMATERIALS
PROMOTE DEVELOPMENT OF FUNCTIONAL TISSUE
ENGINEERING PLATFORMS
Semino CE (1,2)
1. Bioengineering Department, Institut Químic de Sarrià,
Universitat Ramon Llull, Barcelona, Spain; 2. Translational
Centre for Regenerative Medicine, Leipzig University,
Leipzig, Germany
In our laboratory we have been studied several in vitro
3D-tissue systems using nanostructured materials such as
self-assembling peptides scaffolds and natural collagen
gels with the main aim of understanding the basic
biological and biophysical parameters that affect
processes like cell differentiation and function. In my
presentation I will like to review our main results from
experiments using cell lines, tissue and organ derived
cells, including functional mature cells and stem cells,
from species such as human, rat and mouse. I will mainly
focus on liver stem cell differentiation, mature
hepatocyte phenotype maintenance as well as embryonic
and adult fibroblast culture and multi-potent lineage
commitment. The use of soft nanostructured materials
has a clear promising future for applications in tissue
engineering and regenerative therapy.
(17.O1) DIFFERENTIATION OF PRE-OSTEOBLAST CELLS
ON POLY(ETHYLENE TEREPHTHALATE) GRAFTED WITH
RGD AND/OR BMPS MIMETIC PEPTIDES
Zouani OF (1), Chanseau C (1), Durrieu MC (1)
1. INSERM, U1026, Univ. Victor Segalen Bordeaux 2, 146
rue Léo Saignat, 33076 Bordeaux Cedex, France
Introduction. Some BMPs such as BMP-2, BMP-7 and
BMP-9 play a major role in the bone and cartilage
formation (1). After having designed a mimetic peptide of
these growth factors, we immobilized these peptides as
well as a peptide of adhesion (RGD) on polyethylene
terephthalate (PET) surfaces and we evaluated the state
of differentiation of pre-osteoblastic cells. The behavior
of these cells on various functionalized surfaces
highlighted the activity of the mimetic peptides
immobilized on surfaces. The induced cells (observed in
the case of surfaces grafted with BMPs mimetic peptides)
were characterized on several levels. These induced cells
take a different morphology compared to the cells in a
state of proliferation or in a state of extracellular matrix
(ECM) production.
Materials and methods. Mimetic peptides design: The
FATCAT (2) program was used as a tool to search for the
BMP-2 protein homologues. The structural alignment was
performed with STAMP (3) and then optimized with ViTO
(4). The interactions between the three BMPs and the
receptor II were determined according to the
experimental data (5) and also by analyzing the
crystallographic structure of the BMPs in complex with its
receptor II.
Mimetic peptides grafting: PET was grafted in two
subsequent steps (6).
Cell culture: For our study MC3T3-E1 cells, a clonal preosteoblast-like cell line, were cultured in a αMEM
supplemented with 10% serum. The messenger RNA was
quantified with the Syber green intercalating agent at
different time points. For cells observations, scanning
electron microscope (SEM Hitachi S2500) was used.
Results. The detailed study of the interaction between
the BMPs and their receptors and the following analysis
of the selected peptides’ folding contributed to the
design of three mimetic peptides. We clearly observed an
overexpression of Runx2 in surfaces that have the
mimetic peptide of the BMP-2, BMP-7 and BMP-9. In the
case of surfaces grafted with mimetic peptide of BMPs a
formed ECM was observed. Finally, we observed a much
more significant mineralization on surfaces with BMPs
mimetic peptide than on surfaces oxidized or with RGD
after 5 days.
Conclusion. In our study we propose to mimic the
function of certain BMPs by using small peptides. We
show synergy between two pathways due to the surfaces
bifunctionalized with two mimetic peptides.
References.
[1] Senta H et al. Cytokine Growth Factor Rev. 2009
Jun;20(3):213-22.
[2] Barton GJ. Acta Crystallogr D Biol Crystallogr. 2008
;64(Pt 1):25-32.
[3] Ye Y, Godzik A. Protein Sci. 2004 Jul;13(7):1841-50.
[4] Catherinot V, Labesse G. Bioinformatics. 2004
12;20(18):3694-6.
[5] Yin H, Yeh LC, Hinck AP, Lee JC. J Mol Biol. 2008
18;378(1):191-203.
[6] Zouani OF et al, Biomaterials, 201031(32):8245-53.
Keywords. Surface functionalization, osteoinduction,
BMPs mimetic peptide, biomimetic material
(17.O2) EFFECT OF SUBSTRATE STIFFNESS AND
FIBRONECTIN ACTIVITY ON HMSC DIFFERENTIATION
González-García C (1), Moratal D (1), Oreffo ROC (2),
Dalby MJ (3), Salmerón-Sánchez M (1)
1. Universidad Politécnica de Valencia. Spain; 2. University
of Southampton. UK; 3. University of Glasgow. UK
Cell differentiation can be triggered by the properties and
composition of the extracellular matrix (ECM). Whilst we
know that biomaterials can influence stem cell
differentiation, there is some debate in the literature as
to which material properties trigger specific
differentiations. We hypothesize that the reported
differences may be a consequence of the organization of
ECM proteins adsorbed on the surface. This study
correlates fibronectin (FN) adsorption to human
mesenchymal stem cell (hMSCs) differentiation, on a
family of substrates with tailored stiffness and minute
variations in surface chemistry.
Polymer substrates which consist of a vinyl backbone
chain with side groups - COO(CH2)xCH3 (x=0, 1, and 3)
were prepared. Surfaces were characterized (AFM, water
contact angle, elastic modulus). FN was adsorbed from a
solution of concentration 20 µg/mL. The amount of
adsorbed FN was quantified by western-blotting, and its
distribution on the material surface was directly observed
with AFM. hMSCs were cultured on the FN-coated
substrates in serum-free conditions. Focal adhesions,
actin cytoskeleton formation, and the expression of key
transcription factors in the differentiation to bone and
cartilage lineages (such as RUNX2, phospho-RUNX2, and
SOX9) as well as other non-collagenous proteins present
in bone ECM (osteopontin, osteocalcin) were quantified
on the different surfaces by immunofluorescence
followed by image analysis.
Substrates with decreasing stiffness were obtained by
subtle variations in material chemistry, i.e. by sequentially
adding methyl groups in the side group of a vinyl chain.
The same FN density was obtained on every substrate,
but the supramolecular organisation of the protein at the
material interface was different for x=0 than the other
surfaces. This allows one to investigate the effect of
substrate stiffness on cell differentiation after differences
in FN activity have been ruled out. hMSC differentiation
to cartilage and bone lineages depended of the interplay
between substrate stiffness and FN activity.
Keywords. Material properties, protein adsorption, stem
cells differentiation
(17.O3) SELF-ASSEMBLING BIOMIMETIC MATRICES:
OPPORTUNITIES FOR RESEARCH AND THERAPIES IN SKIN
REGENERATION
Ferreira DS (1), Marques AP (1), Reis RL (1), Azevedo HS
(1)
1. 3B’s Research Group - Biomaterials, Biodegradables
and Biomimetics, University of Minho. Portugal
Extracellular matrix (ECM) plays a key role in wound
healing as ECM components are known to have the ability
to regulate cellular processes, such as adhesion, growth
and migration in the different phases of the healing
process. Therefore, bioactive matrices that can mimic
multiple aspects of native cellular environments
(biochemical and physical signals) would be of great
benefit in skin regeneration strategies. Towards this
challenge, we report here the development and
characterization of bioactive membranes that result from
the instant self-assembly between peptide amphiphiles
and the glycosaminoglycan hyaluronic acid (HA), a major
component of skin ECM. To foster cell adhesion and
proliferation on the self-assembling membranes, the
fibronectin-derived RGDS epitope was incorporated into
the peptide structure. Due to their ability to recapitulate
biochemical signals of skin tissue niche, these molecules
offer many unique advantages as starting materials for
skin regeneration applications. Degradation studies
showed that these matrices are susceptible to enzymatic
degradation by hyaluronidase (HAase). In the presence of
HAase at physiological concentration the matrices
degrade gradually over time, which may present an
advantage over other systems, since slow degradation
will induce the migration of cells. The matrix degradation
also provides space that is essential for new tissue
formation. When grown on membranes without the cell
recognition epitope RGDS, human dermal fibroblasts
showed lower adhesion to the matrices when compared
to the ones containing RGDS. We expect that the
proposed biodegradable hybrid matrices could offer
significant potential in skin regeneration strategies, as a
bioactive supportive matrix for promoting wound healing,
and also as model systems for fundamental mechanistic
studies in wound remodeling.
Acknowledgesments. Daniela S. Ferreira acknowledges
the financial support received from Fundação para a
Ciência
e
a
Tecnologia
(PhD
scholarship
SFRH/BD/44977/2008
Keywords. Skin regeneration, biomimetic membranes,
hyaluronan, self-assembly, RGDS, cell adhesion
(17.O4) EFFECT OF 3D-MICRO-ENVIRONMENT ON MICE
CELLS’ GENE EXPRESSION
Fernández-Muiños MT (1), Semino CE (1)
1. Tissue Engineering Laboratory, Bioengineering
Department, Institut Químic de Sarrià, Universidad Ramón
Llull, Barcelona, Spain
Nowadays, the development of biomaterials that
substitute natural cell environments is one of the main
objectives of tissue engineering. Since cells in vivo are in
tridimensional (3D) environments, it is important to
reproduce this condition in vitro for being more adequate
than traditional two-dimensional (2D) cultures. In the
present work we evaluated the influence of a noninstructive soft nanofiber scaffold (RAD16-I selfassembling peptide scaffold) on mouse embryonic
fibroblasts (MEFs). We found that when MEFs were
cultured using this methodology they dedifferentiate into
a primitive progenitor with mesodermal commitment.
Initially, it was observed the spontaneous up-regulation
of a subset of chondrogenic markers, the transcription
factor Sox9 and two main components of the
chondrocytes extracellular matrix, Collagen II and
cartilage specific proteoglycans. Then, we also found that
the expression of the organizer gene noggin (present
during embryogenesis) was also up-regulated very early
in our system only under low matrix stiffness (G’ ~ 100
Pa). This could indicate that the 3D-construct is
undergoing a process that recreates some aspects of a
vestigial
cellular
self-organized
system
during
development. Thus, presenting the subset of early gene
programs to progress autonomously into a default
cartilaginous commitment. Interestingly, none of these
processes occurred to MEFs cultured on 2D. These results
suggest that 3D-environments are more adequate for
natural cell development than the 2D ones, indicating
that self-assembling peptides are promising materials to
be used in cartilage repair.
Keywords.
3D-microenvironments,
Differentiation,
Chondrogenesis
(17.O5) MATRIX ENGINEERING TO LOCALLY CONTROL
CELL FUNCTION AND FORMATION OF ARTIFICIAL
TISSUES
Sala A (1), Lienemann P (2), Kiveliö A (2), Metzger S (2),
Lutolf MP (3), Ehrbar M (2), Vörös J (4), Weber F (2)
1. Eidgenössische Technische Hochschule (ETH) Zürich and
University Hospital Zürich. Switzerland; 2. University
Hospital Zürich. Switzerland; 3. École Polytechnique
Fédérale de Lausanne (EPFL). Switzerland; 4.
Eidgenössische Technische Hochschule (ETH) Zürich.
Switzerland
Introduction. Naturally derived matrices such as collagen
or fibrin have been widely used for 3D cell culture. One
major limitation of these hydrogel materials is their
limited flexibility in engineering applications. We describe
completely synthetic matrices that are modularly
designed and can be specifically tailored towards
biological applications. These materials are based on
biologically inert star-shaped Poly(ethylene glycol). In
order to obtain biological functionality the materials can
be made sensitive towards proteolytic digestion and
decorated with specific integrin ligand domains or
covalently immobilized morphogenetic cues that might
direct cell behaviour2.
Methods. PEG precursors that are decorated with factor
XIII (FXIII) substrate sequences TG (NQEQVSPL) or Lys
(FKG) are reacted in presence of growth factors or cells by
the addition of FXIIIa. The hydrogels were formed in
presence of cells, growth factors and integrin ligand RGD
by casting them into defined layers and by printing. The
building blocks were formed in consecutive steps to reach
a controlled three dimensional organisation of materials
and cells.
Results. We show that by matrix engineering migration of
encapsulated cells can be enabled or prohibited. In
consequence the cellular distribution and movement in
vitro and in vivo can be influenced by matrix design. As
our matrices allow the local presentation of growth
factors, they can not only be used to control the
formation of cellular structures, but also provide them
with instructive cues. We show the 3D arrangement of
cells, matrices and growth factors by means of layer-bylayer assembly or printing. Examples of how cell
behaviour can be controlled locally and how this
translates to tissue-like constructs will be presented.
Discussion. We believe that our novel materials platform
can serve to study fundamental cellular processes in a 3D
setting and to generate tissue mimicking structures.
Acknowledgements. This work was supported by CCMX
and SNF grant CR32I3_125426/1
Keywords. Artificial extracellular matrices; instructive
microenvironments; artificial tissue-like models; printing;
layer-by-layer
(17.O6) INFLUENCE OF CELL DENSITY ON VIABILITY AND
GROWTH OF HUMAN PERIOSTEUM DERIVED CELLS IN
POLYETHYLENE GLYCOL HYDROGELS
Demol J (1), Rizzi SC (2), Van Oosterwyck H (1), Schrooten
J (1)
1. Katholieke Universiteit Leuven. Belgium; 2.QGel SA.
Lausanne . Switzerland
Introduction. Providing cells with a biomimetic 3D culture
environment is key in tissue engineering. However,
determining the initial cell seeding density remains a
pragmatic issue that can determine cell responses and
eventually matrix formation during in vitro culture. This
study explored if viability, metabolic activity and growth
of human periosteum derived cells (hPDCs) alters when
seeded at different densities in biomimetic poly(ethylene
glycol) (PEG) hydrogels.
Methods. After expansion, hPDCs were encapsulated in
30 µl MMP-degradable PEG with RGD motifs (QGel) at
densities of 1 million cells/ml, 2 million cells/ml and 5
million cells/ml. The gels were transferred to 24-well
plates and cultured in growth medium (DMEM + 10% FBS)
for one week. Cell viability was analysed using LIVE/DEAD
staining (Invitrogen). Metabolic activity was monitored
with Alamar Blue (Invitrogen) and changes in cell number
were quantified by measuring DNA content (Invitrogen).
Results. After encapsulation, predominantly viable cells
were found homogeneously distributed throughout the
gel for each cell density. However, at day 7 viability
reduced with increasing cell density. While 77% of the
cells were viable in gels with initial cell density of 1 million
cells/ml, only 41% were alive with the highest initial cell
density. DNA content did not change significantly after
one week, except for gels with 5 million cells/ml. In these
gels, a reduction in DNA content of 40% was measured.
On the other hand, Alamar Blue assay indicated a
significant increase in total cellular activity in gels with 1
million cells/ml (219% increase at day 8 as compared to
day 1).
Conclusions. Significant differences were found between
hPDCs that were 3D cultured in PEG hydrogels at different
seeding densities. These results emphasise that besides a
suitable biomaterial, an optimised initial cell density is
required to obtain the desired biological response.
Keywords. Human periosteum derived cells, cell density,
polyethylene glycol, biomimetic
Figure. Relative cell growth at day 8 (n=3).
(17.O7) BIOACTIVE COMPOSITE SCAFFOLDS MIMIC
BONE TISSUE
Gentile P (1), Mattioli-Belmonte M (2), Baino F (3), TondaTuro C (1), Chiono V (1), Vitale-Brovarone C (3), Ciardelli G
(1)
1. Politecnico di Torino, Department of Mechanics, Italy;
2. Università Politecnica delle Marche, Department of
Molecular Pathology. Italy; 3. Politecnico di Torino, Dept.
of Materials Science and Chemical Engineering. Italy
Introduction. Composites based on apatites and natural
polymers have received increasing attention in bone
tissue engineering due to their ability to preserve the
structural and biological functions of the damaged hard
tissues in a biomimetic way. In this work porous scaffolds,
containing bioactive glass (CEL2) stimulating the
biomineralization and chitosan/gelatin (CH-G) blends
supporting cell adhesion and proliferation, were
developed.
Methods. A 3% (wt/v) CH-G solution (1:2 weight ratio)
was dissolved in 0.5M acetic acid at 40°C. CEL2 was added
to the polymeric solution (POL) to obtain CEL2/POL
composites with various weight ratios between the
components: 0/100; 40/60; 70/30 (wt/wt), coded as
CEL2/POL_0/100; CEL2/POL_40/60; CEL2/POL_70/30.
Genipin was added at defined weight percentage (2.5%
wt/wt). Each mixture was kept at 50°C under stirring,
poured into Petri dishes and freeze-dried at -20°C for 24h.
The obtained scaffolds were characterized for their
morphology, water stability, bioactivity in Simulated Body
Fluid (SBF), mechanical and biological behaviour using
periosteal progenitor cells (PCs)
Results. The increase of CEL2 amount stabilized the
composites in water solutions, as shown by swelling tests.
CEL2/POL samples showed interconnected pores having
an average diameter ranging from 120±5μm for
CEL2/POL_0/100 to 95±5 μm for CEL2/POL_70/30. The
compressive modulus increased by increasing CEL2
amount up to 2.6± 0.3MPa for CEL2/POL_70/30. The SBF
tests showed the high bioactivity of the scaffolds
containing CEL2. MTT viability test using PCs showed the
biocompatibility of scaffolds. Scaffold composition
affected cell morphology (Fig 1).
Conclusions. Composite porous scaffolds containing CEL2
showed an interconnected porosity, bioactivity and
suitable mechanical properties. Moreover, their ability to
sustain PCs growth strengthened the hypothesis of
periosteum as stem cell source for osteo-chondral tissue
regeneration based on in situ cell recruitment.
Acknowledgements. This work was supported by ACTIVE
(Regional Project, Regione Piemonte) and FIRB projects
(RBAP10MLK7).
Keywords. Biomimetic, bioactivity, bone repair,
composite, scaffold, stem cells
(17.O8) THE EFFECT OF NANOFIBER TOPOGRAPHY ON
CELLULAR BEHAVIORS OF PRIMARY RAT ASTROCYTES IN
VITRO
Cao H (1), Marcy G (2), Goh ELK (2), Wang J (3), Chew SY
(1)
1. Nanyang Technological University. Singapore; 2. DUKENUS Graduate Medical School. Singapore; 3. University of
Science and Technology of China
Astrocytes play an important role in the regeneration of
the central nervous system (CNS). In particular, glial scar
formation by reactive astrocytes after nerve injuries
serves as a major hindrance to axonal regeneration.
Unfortunately, detailed understanding on astrocyte
behavior and interaction with microenvironmental signals
remain limited. By investigating astrocyte responses
towards topographical cues, we aim to gain insights to
scaffold design for CNS regeneration. Poly(caprolactoneco-ethyle ethylene phosphate) (PCLEEP) nanofibers with
uniform diameter of 655 ± 11 nm were fabricated by
electrospinning and PCLEEP films were prepared by
solvent-casting as a two-dimensional (2D) control for
primary rat astrocyte culture. Cell proliferation rate as
measured by EdU assay indicated significantly lower EdU
incorporation rate on nanofibers than on films (18.9% vs.
40.2%, p < 0.05). Meanwhile, TUNEL assay demonstrated
a higher apoptosis rate on fibers than on films (11.9% vs.
7.1%, p < 0.05). Astrocytes on nanofibers adopted a
smaller cell area and exhibited an elongated shape as
compared to the fully stretched morphology on films, as
revealed by GFAP immunostaining. However, the
insignificant change in vimentin and GFAP expression
levels as shown by western blotting implied a quiescent
state of astrocytes despite morphological changes in
respond to nanofibers. In our work, the astrocyte
responses including proliferation, apoptosis, cell
morphology and phenotypic changes towards
nanofibrous topography compared to the flat 2D surfaces
was demonstrated. The suppressed growth and enhanced
apoptosis of astrocytes on nanofiber topography suggest
that electrospun nanofiber may serve as a potential biofunctional scaffold for CNS regeneration.
Keywords. Astrocyte, nerve regeneration, nanofiber,
topography, electrospinning
(17.O9) FUNCTIONALLY GRADIENT COLLAGEN/NANOHYDROXYAPATITE OSTEOCHONDRAL SCAFFOLDS
Liu C (1), Dalgarno K (1), Birch M (1), McCaskie A (1)
1. Newcastle University. UK
Introduction. This paper reports a strategy for the
fabrication of functionally gradient collagen/nanohydroxyapatite (HA) composite osteochondral scaffolds
to provide an appropriate physical environment for
hMSCs to migration and differentiation.
Methods. A modified in situ nano-HA precipitation
integrated centrifugation method was used. The resultant
composite plugs were cut into 1 mm think sections
further examinations with respect to its composition,
structure and crystalline. In vitro performance was
evaluated using hMSCs.
Results. The XPS analysis demonstrated that the resultant
composite scaffold has a continuous composition
gradient. It could be categorised into four zones: (1)
superfacial zone consists of pure collagen for
cartilaginous tissue formation; (2) HA-deplete zone with
less than 10% HA content; (3) middle region with HA
content in the range of 10% ~ 50%; (4) distal region with
rich HA content for bone tissue formation. Superfacial
layer exhibited a higher average pore size of 210 mm and
porosity of 80%; HA-deplete layer exhibited a pore size of
160 mm and a porosity of 52%; while as the middle
section of the scaffold shown a pore size and porosity of
123mm and 45%, respectively. The distal region has
dense structure with small pore size in the range of 10 µm
~ 85 µm, and porosity at about 33%.
Histological examination on 4 weeks in vitro cultured
specimens revealed there were patches of bone-like
tissue formed around HA particles at the HA-rich regions;
while patches of cartilage-like tissue were observed with
superficial regions and HA-deplete region.
Conclusions. Such gradient composite scaffold may be an
appropriate substrate that facilitates formation of tissue
for regions of tissue attached to each other, where each
region differs in terms of its resident cell type and
composite, and could lead to a better understanding of
the cellular requirements for co-culture of tissues.
Keywords. Osteochondral tissue engineering, scaffold,
collagen, hydroxyapatite
(17.O10) CROSSLINKED GELATIN NANOFIBRE SCAFFOLDS
FOR PERIPHERAL NERVE TISSUE ENGINEERING
Tonda Turo C (1), Chiono V (1), Gentile P (1), Gnavi S (1),
Cipriano E (2), Zanetti M (2), Perroteau I (1), Ciardelli G (3)
1. Department Of Human and Animal Biology, University
of Turin. Italy; 2. Nanostructured Interfaces and Surfaces
(NIS) Centre of Excellence, Department of Chemistry IFM,
University of Turin. Italy; 3. Department of Mechanics Politecnico di Torino. Italy
Introduction. Fibrous matrices mimic the complex
biological structure of the extracellular matrix and
provide the mechanical support to allow the cells of the
damaged tissue to adhere, proliferate and migrate
properly, forming three-dimensional tissue structures. In
this work, electrospinning was used to prepare γglycidoxypropyltrimethoxysilane (GPTMS) crosslinked
gelatin (GL) nanofibrous scaffolds (GL/GPTMS_NF). The
GL based nanofibrous scaffolds were found to be suitable
matrices for cell attachment and proliferation and
promising materials for peripheral nerve regeneration.
Methods. GL was dissolved in demineralised water at
50°C. Then, GPTMS was added to GL solutions with
various concentrations (the amount of GPTMS was
calculated respect to the molar concentration of amino
groups of hydroxylysine, lysine and arginine residues to
obtain a ratio of 2/1 between the amino groups and the
GPTMS molecules) and then solutions were left stirring
one hour before spinning.
The electrospinning process parameters and solution
concentration were optimized to obtain GL/GPTMS_NF,
which were characterized for their morphology, porosity
and water stability. The cellular response using neonatal
olfactory bulb ensheathing cells (NOBECs) was evaluated
on GL/GPTMS_NF.
Results. The electrospinning parameters (solution
concentration, applied voltage, needle-collector distance,
solution flow rate, temperature) were fixed respectively
at 15% wt./vol., 30kV, 15cm, 10µl/min, 50°C to obtain
fibres with 356±59 nm size (figure 1). NOBECs adhered
and proliferated on the fibrous scaffolds and were found
to align into the fibre direction. Moreover, no apoptotic
cells were found on the fibrous matrices using a DeadEnd
Fluorimetric Tunel System.
Conclusions. Process parameters for the preparation of
GL/GPTMS_NF from aqueous solutions were optimised.
Nanofibers were found to support the in vitro adhesion,
survival and proliferation of glial-like cells.
Acknowledgements. This work was supported by MOVAG
(Compagnia San Paolo) and ACTIVE (Regional Project,
Regione Piemonte) projects.
Keywords. Electrospinning, gelatin, peripheral nerve
tissue engineering
(17.O11) AFM INSIGHTS ON FIBRONECTIN BEHAVIOR AT
THE CELL-MATERIAL INTERFACE
González-García C (1), Salmerón-Sánchez M (2)
1. Center for Biomaterials and Tissue Engineering,
Universidad Politécnica de Valencia, Valencia, Spain;
2. Center for Biomaterials and Tissue Engineering, UPV,
Spain; CIBER-BBN, Valencia, Spain; Centro de
Investigación Príncipe Felipe, Valencia, Spain
Introduction. The initial cellular events that take place at
the biomaterials interface mimic to a certain extent the
natural adhesive interaction of cells with the extracellular
matrix (ECM). In fact, cells cannot interact directly with
foreign materials, but they attach to the adsorbed layer
of proteins. Among the ECM proteins, the importance of
fibronectin (FN) as a mediator of cell adhesion to a
substrate was early recognized. Atomic force microscopy
(AFM) is a powerful tool widely used to analyze biological
molecules, in particular, protein adsorption on material
surfaces, mostly in air environments. Nevertheless, the
systematic investigation of the cell-material interface has
been scarcely addressed in the literature by AFM. This
work investigates fibronectin behavior at the cell-material
interface at the nanoscale making use of AFM.
Additionally, it approaches conditions in which ECM
proteins and cells are found in physiological fluids in vivo
or in culture medium in vitro, working in liquid
environment.
Methods.
Polymers
with
well-characterized
physicochemical properties were used: Poly(L-lactide)
acid,PLLA, Poly(methyl acrylate),PMA, and poly(ethyl
acrylate),PEA. Thin films were prepared by spin-casting
from different polymer solutions on glass coverslips. FN
adsorption on the surfaces was performed from solutions
of different concentrations (5 and 20μg/ml). Osteoblastlike cells and fibroblasts were cultured on the previously
FN-coated substrates in serum-free conditions. Initial FN
conformation on the different surfaces as well as the
cellular reorganization of the FN layer was directly
observed by AFM.
Results and conclusions. Different FN conformations
were obtained for each material. Non-connected FN
aggregates are observed on PLLA and PMA, whereas PEA
is able to induce the formation of a protein network –FN
fibrillogenesis- establishing FN-FN interactions. FN
reorganization was studied visualizing the FN near and far
from cells, in order to compare the effect of cells on
adsorbed FN layer.
Acknowledgments. This work was supported by
MAT2009-14440-C02-01
Keywords. AFM, cell-protein-material-interaction, protein
conformation, FN reorganization
Figure. AFM imaging of cell-mediated FN reorganization
(17.O13) BIOCOMPATIBILITY EVALUATION OF DIFFERENT
BIO-INSPIRED SiC AND ITS UTILITY AS IMPLANTABLE
DEVICES FOR PREVENTING STAPHYLOCOCCUS AUREUS
INFECTIONS
Díaz-Rodríguez P (1), Landín M (1), Couceiro R (2),
Couceiro J (3), González P (4), Serra J (4), López-Álvarez M
(4), León B (4)
1. Dpto. Farmacia y Tecnología Farmacéutica. Fac.
Farmacia. Univ. Santiago. Spain; 2. Instituto de Cerámica.
Univ. Santiago, Spain; 3. Instituto de Ortopedia y Banco
de Tejidos musculoesqueléticos. Univ. Santiago. Spain; 4.
Dpto. Física Aplicada. E.T.S.E. Industriais. Univ. Vigo. Spain
Bio-inspired silicon carbide (BioSiC) is a new ceramic
material obtained from natural resources with excellent
mechanical properties, suitable for bone implants. Its
microstructure has cell-friendly porosity which also allows
the inclusion of therapeutic molecules adequate for
prophylaxis and treatment of device-related infections
due to bacterial adhesion and subsequent bio-film
formation at the implantation site. VEGF can be also
included in order to induce angiogenesis, key process in
tissue engineering.
Disks (ø 6mm x 2mm) of BioSiC obtained from different
woods: pine (Pinus pinnaster), sapelli (Entandrophragma
cylindricum) and oak (Quercus rubra) were produced. The
in vitro biocompatibility was tested with a human
osteoblast cell line (SAOS-2). Cell proliferation on BioSiC
disks unloaded and loaded with vancomycin (1,257 mg),
were compared at different times after seeding using
SEM and also quantified by a MTT assay. The vancomycin
release kinetics were obtained by placing the dried drugloaded disks in vials with 3ml of phosphate buffer (PBS) at
37ºC. The antimicrobiological activity was carried out
against Staphylococcus aureus. Disks were loaded with
VEGF, cultured with Human Mesenchymal cells and
Smooth Muscle cells.
Differences in the BioSiC porosity cause also variations in
the release kinetics. All BioSiCs are characterized by a
rapid delivery during the first 2 h, after which the release
rate decreases.µAccording to the SEM micrographs, the
cells were able to adhere and grow on the BioSiC
surfaces. After 15 days all the surface was covered with
cells which also grow inside the pores. Differences in
microstructure lead variations in cell response. The
highest proliferation was obtained on BioSiC from oak
possibly as a result of the presence of big pores (100
The results validate BioSiC as potential Easy-to-obtain
scaffolds for tissue engineering.
Keywords. BioSiC, microstructure, cell biocompatibility,
device-related infections
(17.O14) DYNAMIC CULTURE OF ENDOTHELIAL CELLS ON
NEW BIOFUNCTIONALIZED 3D-PRINTABLE POLYMERS
FOR SMALL DIAMETER GRAFTS
Novosel EC (1), Klechowitz N (2), Fischer A (2), Meyer W
(3), Schuh C (1), Borchers K (2), Wegener M (3), Krüger H
(3), Walles H (2), Hirth T (2), Tovar GEM (2), Kluger PJ (2)
1. Institute for Interfacial Engineering IGVT, University of
Stuttgart. Germany; 2. Fraunhofer-Institute for Interfacial
Engineering and Biotechnology IGB. Germany; 3.
Fraunhofer Institute for Applied Polymer Research IAP.
Germany
Introduction. Effective vascularization is the central
demand in tissue engineering. Therefore we developed
artificial three-dimensional (3D) blood vessels, which
could be dynamically cultivated to supply surrounding
scaffolds in vitro. New printable polymers were
synthesized, with biomimetic molecules functionalized
and in cell culture experiments analyzed for their
applicability with endothelial cells. The small diameter
vessels were seeded with cells and dynamically cultivated
in a new developed bioreactor system.
Materials and Methods. Different types of alpha, omegahydroxyoligoethers adapted to the needs of rapid
prototyping process have been synthesized and
characterized. The surfaces were biofunctionalized with
covalently immobilized thioheparin and the adhesion
peptide RGDC. The amount and stability of the surface
modification molecules were quantified by XPS, toluidine-
blue assay and ELISA. Adhesion, morphology,
proliferation and functionality of primary human
endothelial cells (EC) seeded on the functionalized
substrates were characterized by viability assays and
immunohistochemistry. Concurrent with ongoing cellmaterial-interaction studies, a bioreactor was developed
for the dynamic culture of ECs in the printed artificial
blood vessels.
Results. The synthesis of suitable polymers for rapid
prototyping processes with remaining functional groups
was successful. Biofunctionalization of the polymer was
shown by the increase in surface sulphate content after
thioheparin immobilization by XPS and photometrical
with toluidin-blue. Seeded endothelial cells on the
functionalized surfaces showed a higher viability and
confluence as the cells on control substrates. All cells
could be characterized using specific EC markers.
Furthermore we designed a bioreactor system for culture
experiments and performed cell experiments under
dynamic flow conditions.
Conclusion. Promising biomaterial research is affected by
the integration of biology, chemistry and engineering. In
our interdisciplinary approach, we developed new 3Dprintable polymers. The materials were successfully
biofunctionalized and cell experiments showed an
increased adhesion of EC on the biomaterial surface.
Acknowledgements. We thank the FraunhoferGesellschaft and the Landesgraduiertenförderung BadenWürttemberg for funding the project.
Keywords. Biofunctionalization, in vitro, tissue
engineering, vascularization, heparin, RGD, rapid
prototyping, small diameter grafts, bioreactor
(17.O15) CHARACTERISTICS OF A BIODEGRADABLE
POROUS
PHB/PCL
NERVE
GUIDE
CONDUIT
BIOFUNCTIONALIZED
WITH
STAR-PEG
HEPARIN
HYDROGEL
Hinüber C (1), Vogel R (1), Brünig H (1), Freudenberg U
(1), Werner C (1)
1. Leibniz Institute of Polymer Research. Dresden.
Germany
Alternatively for autologous nerve grafts, artificial nerve
guidance channels are eligible candidates since
dimensionally mismatch, limited supply and second
surgery can be avoided. The basic strategy of axonal
regeneration by tubulisation is the usage of a hollow
structure that is bridging the gap between the two
stumps of a severed nerve. Ideally, the nerve ends are
inserted into a hollow porous tube, which is
functionalized with biomolecular and/or structural cues,
promoting the oriented growth of axons and ensuring the
transport of nutrients and metabolites over a wide
distance. A bioabsorbable material is considered
advantageous, since the material will be degraded and
metabolized with time. Thus, contusion of the
regenerated nerve and a second surgery site can be
prevented.
The latest results towards the fabrication, modification
and characterisation of a nerve guidance channel which
addresses all of the required properties are presented.
Poly(3-hydroxybutyrate) (PHB) is of great relevance for
medical applications due to its natural origin and
biodegradability. However, PHB is an inherently brittle
material. Blended with poly-ε-caprolactone (PCL),
biodegradable, mechanically stable and bendable hollow
fibers can be fabricated in various dimensions by means
of extrusion or melt spinning. The desired porosity can be
adjusted by particular leaching of a sacrificial component,
for instance polyvinylpyrrolidone. Finally, a well chosen
biomolecular functionalization based on a starpolyethylene-glycol heparin hydrogel layer results into a
tunable bio-hybrid structure. The hydrogel layer allows
for a specific load and release rate of neurotrophins that
are necessary for a particular stimulation of the axonal
regeneration process. This bio-hybrid structure is
expected to be advantageous in comparison to
conventional models since it provides desirable
mechanical, structural and biomolecular characteristics
and is regarded to be beneficial as transplant in
regenerative medical therapy as well as scaffold for in
vitro studies.
Keywords. Biodegradable scaffold, nerve regeneration,
biomolecular functionalisation
(17.O16) HYBRID COMPOSITE SCAFFOLD CONSISTED OF
POLYCAPROLACTONE MICROSTRUTS AND ELECTROSPUN
COLLAGEN-NANOFIBERS
Ahn SH (1), Jin G (1), Hong S (1), Lee JS (1), Kim GH (1)
1. Chosun University. Republic of Korea
In recent years, based on CAD/CAM technology, solid
free-form fabrication (SFF) technologies allow to design
by computer both the microscopic and the macroscopic
shape of scaffolds. The use of computer-based
technology to easily fabricate the scaffolds for tissue
engineering is advantageous because it facilitates the
production of complex computer designed architectures.
Unfortunately, the use of fabricated scaffolds, however, is
challenging since applicable materials are limited to
synthetic biopolymers, and the pore structure can be too
large compared to various cells. Those provided low
biophysical and biocompatible properties to the scaffold.
To overcome these problems, we proposed a hybrid
technology, which combines a melt-plotting system (one
of SFFs) with electrospinning processes, to produce a
hierarchical 3D structure consisting of micro-sized
polycaprolactone (PCL) strands and collagen nanofibers.
To improve the cellular behavior on the scaffold, we
adapted collagen nanofibers in PCL strands since the
collagen is the major constructional element of
extracellular
matrix
(ECM)
and
outstanding
biocompatibility and biodegradability. To complete one
layer, the perpendicular PCL strands were plotted first,
and the upper stage connected to an electrospinning
apparatus was moved automatically to the one-layered
strands; then the collagen nanofibers (the diameter of
collagen fiber is 300–700 nm) were electrospun on top.
The layer-by-layer structure of PCL and collagen was
approximately 20 × 20 × 1.5 mm3. To evaluate the
efficiency of cell attachment, proliferation, and
differentiation within the hierarchical scaffolds, we
cultured osteoblasts (MG63) for regeneration of bone.
The hierarchical scaffold exhibited various positive
qualities. In particular, since the collagen is main
component of ECM, the interactions between the cells
and hierarchical scaffolds containing collagen were much
more positive than those between the cells and
conventional 3D PCL scaffolds.
Keywords. Scaffold, Polycaprolactone, collagen
(17.O17) MODULATION OF 3D-CULTURED hMSC
BEHAVIOUR THROUGH
CHANGES
IN MATRIX
PHYSICOCHEMICAL PROPERTIES
Maia FR (1), Fonseca KB (1), Cruz FA (1), Granja PL (1),
Barrias CC (1)
1. INEB/FEUP. Portugal
Introduction. Gel-like microenvironments based on cellinstructive biomaterials are becoming increasingly
relevant not only for regenerative medicine applications,
but also as 3D cell culture models. We previously
reported the modification of RGD-alginate hydrogels with
protease-sensitive domains, and showed that altering the
matrix bio-functionality had a dramatic effect on 3Dcultured hMSCs. In this study, we looked at the possibility
of modulating cell behaviour only by changing the matrix
physicochemical properties, and compared 2D vs 3D cell
response.
Materials and Methods. RGD-alginates with a range of
polymer concentrations (0.5-2.5wt%), but constant
peptide density were prepared. In situ crosslinking was
promoted
by
adding
CaCO3/GDL.
Hydrogels
microstructure (CryoSEM) and mechanical properties
(DMA) were characterized. hMSCs were combined with
gel-precursor solutions before crosslinking to establish
3D-cultures; or seeded in spin-coated films (2D). Cell
behaviour was analysed and matrices (cell-laden, cellfree) were periodically imaged.
Results. Matrices with different microstructures and
pliability were prepared by changing alginate
concentration. Hydrogels presented loose (0.5wt%) or
dense meshes (2.5wt%), and stiffness increased with
polymer concentration. 2D-cultured hMSCs spread (Factin), and proliferated (3H-thymidine) in all formulations;
but 3D cell response was highly dependent on matrix
properties. Cells maintained high viability (live/dead
assay) and metabolic activity (resazurin), but reduced cell
spreading and growth, particularly at higher polymer
concentrations, due to the biophysical hindrance imposed
by the matrix. In looser hydrogels, cells were able to pull
the hydrogel and migrate towards the core, forming
dense aggregates. The diameter of those matrices
decreased concomitantly.
Conclusions. When cultured under 3D-conditions, cells
become physically constrained by the polymeric network
that interferes with several cellular functions, in particular
with spreading, migration and proliferation, as shown
here. The matrix effect is highly dependent on its
physicochemical properties and has to be taken into
consideration when drawing conclusions from 3D-culture
studies.
Acknowledgements. INL for PhD scholarship, FCT for
funding (PTDC/SAU-BEB/101235/2008 and FCOMP-010124-FEDER-010915).
Keywords. Hydrogel, Stem-cells, 3D-cultures
(17.O18)
ELASTIC BIODEGRADABLE FIBRE-MESH
SCAFFOLDS COATED WITH BIOMIMETIC CALCIUM
PHOSPHATE (CAP) LAYERS FOR PROTEIN DELIVERY
Susano M (1), Leonor IB (1), Reis RL (1), Azevedo HS (1)
1. 3B’s Research Group - Biomaterials, Biodegradables
and Biomimetics, Univ. of Minho, Portugal
Introduction. Several studies suggest the possibility of
optimizing scaffold elasticity to control cell behaviour. In
the recapitulation of cellular microenvironments,
scaffolds may play an important role in providing a
platform to influence the perception and response of cells
to substrate mechanics. We present a fibre-mesh scaffold
with elastic properties and drug delivery functions with
potential in tissue engineering applications and also as 3D
cell culture platform for in vitro studies.
Methods. Fibre mesh scaffolds were produced by wetspinning using a starch/poly-ethylene-vinyl alcohol blend.
The compressive mechanical properties of the scaffold
were evaluated and their morphology analysed by
scanning electron microscopy. The scaffolds were coated
with CaP layers using a biomimetic methodology. To
investigate the carrier potential of these coatings for the
delivery of multiple proteins, fluorescently labelled
proteins were incorporated at different stages of the
coating formation. The protein distribution within the
coating was visualized by confocal laser scanning
microscopy and their release profile determined.
Metabolic activity and proliferation of osteoblasts (SaOs2) seeded onto uncoated and coated scaffolds were
assessed by MTS assay and DNA quantification,
respectively.
Results. We were able to fabricate a highly porous and
degradable 3D structure with elastic behaviour in the wet
state. Protein incorporated in the outer coating layers is
released faster, whereas the protein present in the inner
layers shows a more sustained release, thus showing the
carrier potential of the hybrid scaffolds for the controlled
release of proteins that can regulate the function of
seeded cells. MTS and DNA assays proved that the cells
seeded on the scaffolds remain viable with an increased
metabolic activity and proliferation rate.
Conclusions. The structural and degradable properties
and positive cellular response suggest that the developed
fibre-meshes may be good candidates as tissue
engineering scaffolds.
Acknowledgements. M. Susano thanks the Portuguese
Foundation for Science and Technology for providing her
a grant PTDC/CTM/67560/2006.
Keywords. Biodegradable scaffolds, elastic behaviour,
biomimetic CaP coating, protein delivery
(17.O19) RESORBABLE CALCIUM PHOSPHATE SCAFFOLDS
FOR BONE REPAIR AND REGENERATION
Newe C (1), Cunningham E (1), Buchanan F (1), Walker G
(2), Prendergast P (3), Lennon A (3), Dunne N (1)
1. School of Mechanical and Aerospace Engineering,
Queen’s University Belfast, UK; 2. School of Chemistry and
Chemical Engineering, Queen’s University Belfast, UK; 3.
Trinity Centre for Bioengineering, Trinity College, Dublin 2,
Ireland
Introduction. Development of tissue-engineered scaffolds
for bone repair and regeneration which deliver results
equivalent to autografts remains a clinical imperative.
Hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP)
are widely used due to their excellent biocompatibility
and osteoconductivity. However, differences in their
respective resorption rates present difficulties in
achieving optimal tissue formation. Developing a biphasic
HA:β-TCP scaffold could potentially deliver the optimum
resorption rate. The aim of this study was to understand
the material factors influencing the rate of resorption of
biphasic scaffolds.
Methods. Porous scaffolds of five HA:β-TCP ratios (0:100,
25:75, 50:50, 75:25, 100:0wt%) were fabricated using a
sponge replication technique with natural marine sponge
(Spongia agaricina) as the precursor. Solid HA:β-TCP
tablets were also produced for comparison. An in-vitro
model (pH4 buffer solution, 37degC) was used to simulate
osteoclast-like resorption. Samples were removed from
solution at intervals of 1, 6, 24, 48, 72, 96 and 120h.
Gravimetric and dimensional analysis was conducted.
Micro-computed tomography scans were conducted to
determine changes in scaffold architecture as a function
of time in solution.
Results. Gravimetric analysis (Figure1) showed an
increase (R2=0.8056-0.9469) in mass loss over time. A
significant difference (p-value<0.001) was observed for
the porous scaffolds when compared to the solid tablets;
due to the greater exposed surface area of the scaffolds.
Increasing the β-TCP content in the ceramic slurry
resulted in a proportional increase (R2=0.2153-0.7070) in
mass loss.
Conclusion. Scaffold resorption rate can be controlled by
combining HA and β-TCP in scaffold fabrication; and
establishing an appropriate mix-ratio could optimise
resorption in line with bone remodelling. The
experimental data will be used to validate a dissolution
algorithm
for
computational
mechano-biology
simulations of tissue differentiation, thereby helping
maximise scaffold properties and, potentially, reduce the
need for ‘trial and error’ research.
Acknowledgements. This work is part-funded by Science
Foundation Ireland (North-South Supplement Grant).
Keywords. Calcium phosphate, Dissolution, Scaffolds
require biomaterial scaffolds that can handle dynamic
loading. Photo Fenton polymerized resilin-CBD and
cellulose nano-composite systems may offer the
biomechanical properties of resilience, toughness and the
durability necessary for load bearing orthopaedic
implants. We are currently investigating the combination
of plant derived human collagen with resilin composites
to support cell proliferation and neo-tissue formation.
Keywords. Resilin, tissue engineering, composites
A
(17.O20) BIOINSPIRED NANOCOMPOSITES OF RESILIN
AND CELLULOSE WHISKERS FOR TISSUE ENGINEERING
APPLICATIONS
Dgany O (1), Lapidot S (2), Rivkin A (2), Sharon S (2), Bella
Arinos S (2), Shoseyov O (2), Qin G (3), Hu X (3),Kaplan D
(3)
1. CollPlant Ltd. 3 Sapir St. POB 4132 Nes Tziona, 74140,
Israel; 2. The Robert H. Smith Institute of Plant Sciences
and Genetics in Agriculture, The Robert H. Smith Faculty
of Agriculture, Food and Environment, The Hebrew
University of Jerusale. Israel ; 3. Department of Biomedical
Engineering, 4 Colby Street, Tufts University, Medford,
Massachusetts 02155. USA
Introduction. Resilin is an elastomeric rubber-like protein
secreted by insects to specialized cuticle regions, in areas
where high resilience is required. Resilin binds to the
cuticle polysaccharide chitin via a chitin binding domain
and is further polymerized through oxidation of the
tyrosine residues resulting in the formation of dityrosine
bridges and assembly of a high-performance proteincarbohydrate composite material combining the elasticity
of resilin and toughness of chitin. Inspired by the
remarkable mechanical properties of insect cuticles we
hypothesized that novel composites of cellulose and
resilin, cross linked via non toxic resilin polymerization
chemistry will enable the fabrication of implants with
suitable mechanical properties for orthopaedic load
bearing implants.
Materials and Methods. Recombinant resilin fused to
cellulose binding domain (CBD) was expressed and
purified from E. coli. Cellulose Whiskers (CW) were
prepared by H2SO4 hydrolysis of micro crystalline
cellulose followed by sonication resulting in honey like
liquid crystal suspensions. CW suspensions were
subsequently cast into aluminium molds and lyophilized
resulting in highly porous sponges. Resilin-CBD solutions
were embedded into the sponges and polymerized by
ruthenium-bis -pyridinium (Fig. 1B) or the Fe/H2O2
photo-Fenton system (Fig. 1C, 1D) that we have recently
developed for this protein.
Results. Composite sponges of resilin and CW resulted in
dramatic alteration in mechanical behaviour including
high elasticity and no plastic deformation/high resilience
following repeated cycles of mechanical stress (Fig. 1B). In
addition we report the successful polymerization of resilin
by photo-Fenton reaction resulting in elastic, rubber-like,
hydrogels (Fig. 1C,1D).
Discussion and Conclusions. Tendons, ligaments and
spine related diseases are among the most common
health problems in adult populations. In spite of
impressive advances in regeneration, these tissues
B
C
D
Fig. 1. A; Pure low resilience CW sponge B;
Highly elastic composite resilin- CBD/CW
sponge. C; Acid hydrolysis products of photoFenton polymerized resilin samples separated
on a C-18 reverse phase column with
fluorescence analysis for di-tyrosine detection
D; Novel photo-Fenton polymerized elastic
resilin
(17.O21) DEVELOPMENT OF BIOACTIVE MEMBRANE
SCAFFOLDS FOR TISSUE ENGINEERING
Tejeda-Montes E (1), Mata A (1), Smith KH (1), LópezBosque MJ (1), Poch M (1), Engel E (2), Alonso M (3)
1. The Nanotechnology Platform, Parc Cientific Barcelona.
Spain; 2. Institut de Bioenginyeria de Catalunya, IBEC
Barcelona. Spain; 3. BIOFORGE Group, University of
Valladolid. Spain
Introduction. The use of thin bioactive membrane
scaffolds in tissue engineering and regenerative medicine
could have many applications in vivo, directly replacing or
stimulating tissues, and in vitro, facilitating wellcontrolled studies of cell-cell communication or nutrient
permeability. In this work we report on the combination
of a top-down/bottom-up approach to develop thin selfsupporting bioactive membranes based on Elastin-Like
Polymers (ELPs).
Materials and Methods. ELPs containing the cell adhesive
epitope arginine-glycine-aspartic acid-serine (RGDS) were
synthesized using standard recombinant protein
production techniques and cross-linked with 1,6hexamethylene-diisocyanate (HMDI). The ELP membranes
were fabricated by a drop-casting/evaporation technique
using a spin-coater to precisely control membrane
thickness. Membranes were fabricated with a variety of
topographical patterns on either one or both sides,
uniform and well-defined through-holes, and exhibiting
multi-layers. Membrane swelling and stiffness were
characterized by atomic force microscopy (AFM),
nanoindentation tests, and scanning electron microscopy
(SEM). The membrane biocompatibility and bioactivity
were assessed by in vitro culture using rat mesenchymal
stem cells (rMSCs).
Results. Membranes were reproducibly fabricated with
thicknesses varying between 500nm–100µm depending
on the fabrication conditions, exhibited sufficient
structural integrity to be handled and sutured, and served
as in vitro cell culture substrates. Membranes were also
fabricated comprising topographical features with heights
ranging between 500nm and up to 10µm. Optical,
inmuno-fluorescence, and scanning electron microscopy
demonstrated that rMSCs adhered on the ELP
membranes, exhibiting a spread morphology and welldefined actin cytoskeleton.
Discussion and Conclusions. We have developed a variety
of fabrication techniques based on micro and
nanotechnologies to create thin self-supporting
membranes that comprise bioactive epitopes and a
variety of topographical, morphological, and structural
components that could be fine-tuned to stimulate specific
biological processes. These structures could potentially
serve as thin bioactive, biomimetic, multifunctional, and
biodegradable scaffolds for a variety of applications in
tissue engineering and regenerative medicine.
Keywords. Membrane scaffolds, microfabrication,
bioactivity, elastin-like polymers
bioactive hydrogels facilitate optimization of bioactivity
and mechanical properties which offers unique control
over the endothelialization of the graft. However, scaffold
properties that promote endothelialization may not be
consistent with the mechanical properties necessary to
withstand physiological loading. To address this issue, we
have reinforced Scl2/PEG hydrogels with an electrospun
polyurethane mesh. This multilayer vascular graft design
decouples requisite mechanical properties from
endotheliazation processes and permits optimization of
both design goals.
Methods. Polyurethane chemistries and electrospinning
parameters were varied to optimize compliance, burst
pressure, and suture retention of composite grafts.
Platelet adhesion under flow of whole blood was
evaluated to determine graft thrombogenicity.
Additionally, endothelial cell (EC) adhesion and migration
was evaluated in response to changes in Scl2
concentration and identity in the hydrogel layer.
Results. Constructs were developed with biomechanical
properties comparable to human saphenous veins in
current clinical use (Table 1). Platelet adhesion was
statistically less than collagen-coated tissue culture
polystyrene and equivalent to PEG hydrogels. EC
adhesion on the Scl2/PEG hydrogels was comparable to
collagen-based hydrogels.
Conclusions. Our multilayer design can achieve a nonthrombogenic intimal layer that promotes EC adhesion
and migration while providing mechanical properties of
current autologous grafts. These results demonstrate the
great potential of these vascular grafts as an off-the-shelf
graft for small diameter arterial prostheses.
Keywords. Collagen-Mimetic Proteins, PEG hydrogels,
Vascular graft
Property
Burst Pressure
Compliance
Suture Retention
Multilayer Graft
1404 ± 40 mmHg
5.2 ± 0.5 mmHg-1X10-4
306 ± 21 gf
Saphenous Vein
1680 ± 307 mmHg
4.7 mmHg-1X10-4
196 ± 2 gf
Table 1. Biomechanical properties of multilayer vascular
grafts are comparable to saphenous vein allografts
currently used in bypass surgeries.
(17.O22) MULTILAYER VASCULAR GRAFTS BASED ON
COLLAGEN-MIMETIC HYDROGELS
Browning MB (1), Dempsey D (1), Guiza V (1), Becerra S
(1), Rivera J (1), Hook M (1), Russell B (1), Clubb F (1),
Miller M (1), Fossum T (1), Hahn M (1), CosgriffHernandez E (1)
1. Biomedical Engineering, Texas A&M University. USA
Introduction. The urgent clinical need for small-caliber
vascular prostheses has prompted investigation of
biomimetic grafts with properties that more closely
match native blood vessels. To this end, we have
established a novel biomaterial platform based on a
collagen-mimetic protein derived from group A
Streptococcus, Scl2.28. Scl2 has the triple helical structure
of collagen, but unlike collagen, Scl2 is a nonthrombogenic protein that can be modified to have
selective cell adhesion. We have developed the
methodology to incorporate Scl2 proteins into a
poly(ethylene glycol) (PEG) based hydrogel matrix. These
(17.O24) INTEGRATION OF MULTIPLE CELL-MATRIX
INTERACTIONS INTO ALGINATE SCAFFOLDS FOR
PROMOTING CARDIAC TISSUE REGENERATION
Sapir Y (1), Kryukov O (1), Cohen S (1)
1. Avram and Stella Goldstein-Goren Department of
Biotechnology Engineering, Ben-Gurion University of the
Negev, Beer-Sheva, 84105, Israel
Background and aims. Engineering a functional cardiac
tissue in vitro is one of the most challenging tasks for
tissue engineers. In this research, we aimed to
reconstruct the microenvironment promoting cardiac
tissue regeneration by presenting multiple cell-matrix
interactions, in a similar manner to their presentation by
the extra-cellular matrix (ECM) in vivo. Thus, two
fibronectin-derived peptides, (RGD and heparin binding
peptide (HBP), were bound to alginate scaffold,
mimicking the specific interactions of ECM with integrin
and syndecan on cell membrane, respectively
Methods and Results. The peptides (GGGGRGDY,
GGGGSPPRRARVTY or their combination) were
covalently-attached to alginate via the carbodiimide
chemistry, creating an amide bond between the peptide
terminal amine group and the alginate carboxylic group.
High efficiency of peptide attachment and uniform
distribution in the scaffold were confirmed by using
fluorescently-tagged peptides. Peptide binding did not
have an effect on scaffold internal morphology (e.g.,
porosity by Scanning Electron Microscope) or matrix
stiffness. The HBP/RGD-modified scaffold was more
favorable compared to that with single peptide- or
unmodified alginate scaffolds, as reflected by the
increased AKT phosphorylation, indicating to the
activation of adhesion-dependant pathway and prosurvival signaling. Furthermore, already by day 7, welldeveloped myofibers with distinguished striation were
observed in HBP/RGD scaffolds . In the RGD-attached
scaffold, sporadic islands of striation were seen, but no
developed myofibers. In contrast, the HBP-modified and
unmodified scaffolds had no such as effect on cardiac
reorganization. Finally, alpha-actinin, Connexin-43 and NCadherin expression profiles presented better tissue
maturation and regeneration of a functional cardiac
muscle tissue within the scaffolds with the multiple
functional cues
Conclusions. Our data establish the potential use of
HBP/RGD alginate scaffolds as a better ECM-mimicking
microenvironment for inducing regeneration of functional
cardiac tissue, in vitro.
Keywords. RGD, heparin binding peptide, alginate
scaffold, cardiac tissue regeneration
Figure 1. (a) Confocal images of cardiac constructs in
HBP/RGD, HBP-, RGD-modified and unmodified scaffolds
at different times during cultivation. Cardiac cells are
stained for F-actin(red), sarcomeric alpha-actinin (green)
and nuclei (blue). (b) Expression of representative
cardiomyocyte proteins by Western Blot. The relative
folds of increase in the contractile protein a-sarcomeric
actinin (A), the cell-cell adhesion molecule N-Cadherin of
the intercalated disc (B) and the gap junction protein
Connexin-43 (C).Asterisks denote significant difference (2way ANOVA), when *p<0.05, **p<0.01 and ***p<0.005.
(17.O25) CELL HARVESTING FROM ELASTIN-LIKE
RECOMBINAMERS GRAFFTED SURFACES
Pierna M (1), Santos M (1), Girotti A (1), Arias FJ (1),
Rodríguez-Cabello JC (1)
1. BIOFORGE, CIBER-BBN, University of Valladolid, Spain
This work describes an efficient method for removing
single cells or cell sheet which maintaining intact cell-cell
and cell-extracellular matrix (ECM) interactions without
necessity of using mechanical, physical or enzymatic
methods. For this aim bioactive elastin likerecombinamers (ELRs) were covalently grafted to glass
cover slides or polystyrene creating bioactive thermoresponsive surfaces by Click Chemistry.
The ELRs are a class of proteinaceous materials which
exhibit smart behaviour, bioactivity and unmatched
biocompatibility.The smart behaviour is due to transition
temperature which permit us obtain thermo-responsive
surfaces by grafted these polymers.Their biosynthesis by
means of recombinant DNA technologies provides
tailored polymers with an absolute control of the
architecture, lack of randomness in amino-acid sequence,
stereochemistry, and exact molecular weight.Moreover,
is possible to build complex amino-acid sequences
including different and specific functionalities as adhesion
cellular sequences or to modify the transition
temperature by a single change in their amino-acid
sequences. Each step of the modification of the polymer
and surfaces (glass and polystyrene) before reaction as
well as the polymer functionalized surfaces were
characterized by water contact angle measurements,
amino-acid analyses, mass spectroscopy, infrared
spectroscopic analysis, TOF-SIMS, XPS and AFM.
Thus, we obtain bioactive thermo-responsive surfaces to
achieve detachment single cells or cell sheet without use
mechanical, physical or enzymatic methods which can
damage the interaction between cell-cell and cell-ECM.
For this aimis necessary a single decrease on the
environmental temperature. We also check if these single
cells or the cell sheet detached are viablethroughout
colorimetric methods and flowcytometry. They are able
to adhesion, grow and proliferate to new polystyrene
tissue
culture
surface
after
temperature
treatment.Moreover, it is possibly to combining different
bioactive sequence polymers more or less specific to
create particular cellular areas onto surface building cell
sheet that mimicking natural tissue.
Keywords. Elastin-like Recombinamers, Click Chemistry,
Cell-Harvesting
(17.O26) BIORESORPTION BEHAVIOUR OF ALGA-HA
BONE GRAFT SUBSTITUTES
Walsh P (1), Buchanan F (1), Walker G (1), Clarke S (1)
1. Queen's University Belfast. UK
One area for clear improvements in synthetic grafts is
their in situ bioresorption performance. Clinicians would
prefer the graft to resorb completely to minimise the risk
of failure from infection. From an engineer’s prospective,
resorption should be in synchrony with new bone
formation. Too slow a resorption rate, and new bone
formation will be impeded, whereas too fast a rate will
cause mechanical instability at the defect site. The
bioresorption profile of synthetic calcium phosphate
(CaP) bone grafts depends on the physicochemical
properties of the material involved and the biological
environment: some highly crystalline hydroxyapatite (HA)
bone grafts, for example, have been reported to remain
in situ for up to five years. This study investigated the
bioresorption profile of a semi-crystalline algal-HA (QUB
HA) and a highly crystalline algal-HA (manufactured by
methods analogous to those used to produce the
commercially available Algipore). The materials were
placed in vitro in both a cell free and a cellular
environment, in their original granular form and formed
into discs. For cell-free dissolution the materials were
placed in buffer solutions at pH7.4 and pH4.0 with
agitation at 37°C. Dissolution behaviour was monitored
using inductively coupled plasma-mass spectroscopy (ICPMS) to quantify Ca, P and Mg ions dissolution and mass
loss over 28 days. An osteoclast assay using RAW 264.7
cell line expanded in sRANKL was performed on the
materials at 6 and 12 days. ICP and SEM quantifying
resorptive pit formation were used as an outcome
measure. The results showed that processing conditions
affect the rate of dissolution/bioresorption with respect
to time and both decreased with increased crystallinity
and porosity.
Keywords. Algae, Microporous, Calcium Phosphate,
Bioresorption
(17.O27) IN VITRO BEHAVIOUR OF MESENCHYMAL STEM
CELLS ON A CONDUCTIVE ELECTROSPUN SILK FIBROIN
NANOFIBER SCAFFOLD COATED WITH POLYPYRROLE
FOR BIOMEDICAL APPLICATIONS
Aznar Cervantes SD (1), Meseguer-Olmo L (2), Roca García
MI (3), Cragnolini F (2), Cenis Anadón JL (1), Blanquer
Blanquer M (2), Rodriguez Lozano FJ (2), Fernández Otero
T (2), Moraleda Jimenez JM (2)
1. Department of Biotechnology. Instituto Murciano de
Investigación y Desarrollo Agrario y Alimentario (IMIDA).
Murcia, Spain; 2. Hospital Virgen de la Arrixaca.
Universidad de Murcia. Spain; 3. CEMI, Universidad
Politécnica de Cartagena. Spain
Introduction. The possibilities of scaffolds composed of
nanofibers of silk fibroin (SF) could be greatly enhanced
by conferring them electro conductive functionalities.
Here we present the generation of a hybrid material
made of SF coated with polypyrrole (PPy-SF), studying its
biocompatibility as scaffold for proliferation of primary
human mesenchymal stem cells.
Methods. The silk fibroin mesh was obtained by electro
spinning of a 17% (w/v) SF solution (in HFIP). After the
annealing with methanol the meshes were coated with
polypyrrole. The characterizations of physical, structural
and mechanical properties were developed using SEM,
FTIR spectroscopy, and a mechanical tester.
Electrochemical experiments were also performed.
MSCs were obtained by direct aspiration of iliac crest
from volunteer donors. The cells were isolated by
gradient ficoll using a SEPAX™ System device and
cultivated in DMEM supplemented with 10% FCS and
penicillin/streptomycin. Pieces of mats were seeded (2.0
x 104 cells/cm2) into 24 wells cell culture plates and
proliferation was measured by MTT staining at 1, 7, 14
and 21 days.
Results. The average diameter of SF-PPY coated fibers
was 2630 nm (ranging from 472 to 8670 nm). FTIR
spectroscopy indicates that the conjugated polymer has
some interactions with the peptide linkage affecting to SF
macromolecular chains.
Cells showed an excellent adhesion on the materials
tested just 72h after the seeding and a slight growing
tendency was observed.
Conclusions. Our results show the ability of electrospun
silk matrices to support MSCs attachment, spreading and
growth in vitro. We added an original variable, using
conducting polymers (PPY) adsorbed to SF fibers in order
to increase the electric conductivity of the mats with its
possible additional benefits due to the relevance of
electric fields in cell function and spatial disposition.
Acknowledgements. This work was supported by INIA
and by RETICS RD/0010/2012 grants from the ISCIII.
Keywords. Fibroin, polypyrrole, nanofibers, MSCs
(17.O28) ASSEMBLY OF PLATELET-LYSATE LOADED
CHITOSAN-CHONDROITIN SULFATE NANOPARTICLES AS
NEW THREE-DIMENSIONAL HYDROGEL CONSTRUCT FOR
ENTRAPMENT OF HUMAN ADIPOSE DERIVED STEM
CELLS FOR CARTILAGE TISSUE ENGINEERING
Santo VE (1), Popa EG (1), Gomes ME (1), Mano JF (1),
Reis RL (1)
1. 3Bs Research Group - Biomaterials, Biodegradables and
Biomimetics, University of Minho. Portugal
Introduction. Platelet lysates (PL) are an outstanding
autologous source of growth factors (GFs) that can play
an enhancement role over the proliferation and
differentiation ability of mesenchymal stem cells. Natural
based chitosan/chondroitin sulfate nanoparticles (CH/CS
NPs) were developed with the ultimate goal of
encapsulating bioactive agents to promote and enhance
cartilage regeneration. Previous studies performed in our
group reported the successful incorporation of PL in these
NPs, which were then released in a controlled manner in
two and three dimensional (2D and 3D) in vitro cultures
of human adipose derived stem cells (hASCs), enhancing
the proliferation rate of hASCs while they are
differentiating into the chondrogenic phenotype. The
CH/CS complex mimics the extracellular matrix (ECM)
interactions and when used at determined
concentrations, the PL-loaded NPs can assemble in simple
and quick mode and form a 3D stable hydrogel while in
suspension with hASCs, following a mild centrifugation.
The cells are then entrapped in this enriched 3D
environment, recreating the ECM in cartilage.
Methods. The PL-loaded hydrogels were cultured in vitro
in chondrogenic and basal mediums up to 28 days and
were characterized for cell viability, proliferation,
glycosaminoglycans
production,
histology,
immunohistochemistry and gene expression (by
polymerase chain reaction) for cartilage regeneration.
hASCs pellets and empty NPs hydrogels were used as
controls.
Results. The presence of PLs influences the biological
response of the entrapped cells, stimulating their
viability, proliferation and production of a cartilage ECM
throughout the culture time. It was also possible to detect
an enhancement of gene expression for chondrogenic
markers, indicating the positive role of GFs release from
PL on the differentiation ability of hASCs.
Conclusions. The assembly of PL-loaded NPs in
combination with hASCs enabled the development of an
innovative and effective 3D system with multiple
functionality and thus with high potential for application
in cartilage tissue regeneration.
Acknowledgments. FCT for the PhD grants of Santo VE
and
Popa
EG
(SFRH/BD/39486/2007
and
SFRH/BD/64070/2009), IPS, Hospital da Prelada.
EXPERTISSUES
(NMP3-CT-2004-500283),
Find&Bind
(NMP4-SL-2009-229292).
Keywords. Adipose derived stem cells; platelet lysate;
hydrogel; cartilage tissue engineering
(17.O29) ABILITY OF A MARINE SPONGE-DERIVED
POROUS HA SCAFFOLD TO SUPPORT BONE CELL
GROWTH AND DIFFERENTIATION
Clarke SA (1), Cunningham E (1), Choi SY (1), Dunne N (1),
Walker G (1), Buchanan F (1)
1. Queen's University Belfast, UK
Bone tissue engineering may provide an alternative to
autograft use for particular clinical applications, however
scaffold optimisation is still required to maximize bone
ingrowth. In designing scaffolds, pore size, distribution
and interconnectivity may affect bone cell attachment,
proliferation and differentiation and there is evidence
that cells prefer a degree of non-uniformity and a
structure that closely resembles that of natural bone. The
aim of this study was to compare scaffolds derived from a
porous marine sponge (Spongia agaricina) with those
derived from synthetic polyurethane foam.
Hydroxyapatite scaffolds of 1cm3 were prepared via
ceramic infiltration of marine sponge and a polyurethane
(PU) foam. Porosity, pore size distribution and pore
interconnectivity were measured. For biocompatibility
studies, human foetal osteoblasts were seeded at 1x105
cells/scaffold for up to 14 days. Cytotoxicity, cell number,
morphology and differentiation were investigated. PUderived scaffolds had 84-91% porosity with pore sizes
ranging from 50μm-1000μm (average 577μm) and
99.99% pore interconnectivity. In comparison marine
sponge-derived scaffolds had 56-61% porosity with pore
sizes ranging from 0-500 μm (average 349μm) and 99.9%
pore interconnectivity. hFOB studies showed that more
cells were found on marine sponge-derived scaffolds at
d4, d7 and d14 than on the PU scaffold but there was no
difference in cell differentiation as measured by alkaline
phosphatase activity and expression of cbfa-1, collagen I
and osteocalcin. XRD and ICP showed that more Ca and Si
ions were released from the marine-derived scaffold.
Three dimensional porous constructs have been
manufactured that support cell attachment, proliferation
and differentiation but significantly more cells were seen
on marine-derived scaffolds. This could be due both to
the chemistry and pore architecture of the scaffolds with
optimum mechanical stimulus of the cells derived from
the pore characteristics, in addition to a biological
stimulus from increased dissolution of Ca and Si ions.
Keywords. Marine Sponge, Biomimetic, Scaffold, in vitro
(17.O30) DESIGN OF INDUCTIVE SCAFFOLD FOR THE
OSTEOCHONDRAL DIFFERENTIATION OF HUMAN
MESENCHYMAL STEM CELLS (HMSC)
Re'em T (1), Cohen S (1)
1. Ben-Gurion University. Be'er Sheva, Israel
Challenge and Goals. HMSC differentiation depends on
the environment wherein the cells reside, especially on
the spatio-temporal presentation of the differentiationinductive factors. We aim to reconstruct the
microenvironment
promoting
the
osteochondral
differentiation of MSCs, by presenting the chondroinductive Transforming Growth Factor-beta1 (TGF-beta1),
and the osteo-inductive Bone Morphogenetic Protein-4
(BMP-4) in a similar manner to their presentation by the
extracellular matrix.
Methods and Results. TGF-beta1 or BMP-4 were
individually bound to two alginate-sulfate-containing
macroporous alginate scaffolds, subsequently to be
combined into a bilayered osteochondral inducing
system. The affinity binding to alginate sulfate resulted in
a sustained factor release for 7 days, in contrast to the
burst release of these factors from unmodified scaffolds.
The factors retained their bioactivity, as revealed by the
enhanced collagen deposition in fibroblasts culture.
HMSCs, seeded in these scaffolds, showed prolonged and
elevated phosphorylation levels of Smad2 and ERK1/2, for
up to 14 days, indicating the long-term activity of the
affinity-bound factors. Masson's trichrome staining and
immuno-staining of 14 days-old cell constructs
demonstrated substantial deposition of collagen and
collagen type II, in the TGF-beta1/affinity-bound layer and
the cells in this layer presented round morphology of
committed chondrocytes. In the BMP-4/affinity-bound
layer, elevated levels of alkaline phosphatase (ALP) and
increased mineralized bone matrix deposition after 3
weeks indicated their osteogenic differentiation. The ALP
activity in the bilayered system was significantly greater
compared to the activity in the only BMP4-loaded
scaffolds, suggesting a mutual effect of the factors and
their spatial arrangement within the system on MSC
differentiation.
Conclusions. These data indicate the potential use of the
affinity-binding alginate scaffolds combined with spatial
presentation of TGF-beta1 and BMP-4 for guided
differentiation of hMSCs; allowing the reconstruction of
the microenvironment for osteochondral tissue
formation.
Keywords. Mesenchymal stem cells, osteochondral,
microenvironement, affinity-binding, scaffold
(17.O31) FABRICATION AND CHARACTERISATION OF
ELECTROSPUN, TUBULAR, AXIALLY ORIENTED FIBRILAR
GELATIN
SCAFFOLDS
FOR
VASCULAR
TISSUE
ENGINEERING
Elsayed Y (1), Lekakou C (1), Tomlins P (2)
1. University of Surrey. UK; 2. National Physics Laboratory.
UK
Electrospinning of polymer solutions or melts to develop
fibrous constructs that can be used as cell scaffolds is a
promising fabrication technique for tissue engineering
due to a number of reasons, including controlled fibre
diameter to low nanoscale that can be sometimes useful
for cell adherence, incorporation of nanoparticles or a
second phase to mimic the natural extra cellular matrix
(ECM) as for collagen/elastin vascular grafts, the ability to
control porosity, pore size and fibre orientation with
relative ease coupled with the cost effectiveness of the
process. Gelatin has proved to be an advantageous
material to use in tissue engineering due to its favourable
interaction with the cells, its chemical and hierarchical
physical structure being similar to the collagen structure
and its low cost. However gelatin lacks the mechanical
strength required for the tissue engineering process
leading to the necessity of crosslinking. The following
work discusses the fabrication of electrospun, tubular,
fibrous, gelatin scaffolds with axial fibre orientation
manufactured with a variety of structural features, i.e.
different fibre diameter, fibre volume fraction, degree of
crosslinking, pore size and porosity. Furthermore, tubular
fibrous scaffolds with graded structure and porosity from
the outer layer to the lumen have also been fabricated.
Smooth muscle cells (SMCs) and endothelial cells (ECs)
are then seeded on the scaffolds and monitored for
adherence, mass transfer into the scaffold, growth and
growth of cells layer surrounding each fibre. The cell
proliferation across the depth of the scaffold is examined
by staining. The results show a direct correlation between
the physical properties of the crosslinked scaffolds and
the transfer rate and proliferation rate of the cells. Finally
the cytotoxicity of the glutaraldehyde, used as a
crosslinking agent, is also examined to determine its
effect on the cells.
Keywords. Scaffold, electrospinning, gelatin, smooth
muscle cells
(17.P1) 3D CELL GROWTH IN ALGINATE FOAMS
Andersen T (1), Markussen C (1), Heier-Baardson H (1),
Dornish M (1), Ward C (2), Mullen P (2), Langdon S (2)
1. NovaMatrix, FMC BioPolymer, Sandvika, Norway;
2. Breakthrough Breast Unit, University of Edinburth,
Edinburth, U.K.
Growing cells in a 3-dimensional (3D) matrix instead of
traditional 2D cultures can approximate cell architecture
and cell-cell contact found in tissues, organs and tumors.
NovaMatrix-3D™ is an alginate-based cell culture system
comprising an alginate foam matrix and an alginate
immobilizing solution. In principle, cells are first
suspended in a sodium alginate solution then the cell
suspension is applied to calcium alginate foams. In situ
gelation occurs when calcium ions are donated from the
foam cross-linking the added alginate, effectively
entrapping the cells within the pores throughout the
foam. The utility of this new cell culture system is shown
using NHIK 3025 (cervix carcinoma), MCF7 (breast
adenocarcinoma), ZR-75-1 (breast ductal carcinoma),
C2C12 (myoblast), NIH:3T3 (fibroblast) and NIH:OVCAR-3
(ovarian adenocarcinoma). Cell localization within the
foam was visualized using confocal microscopy to identify
fluorescently labeled cells (CellTrace™ CFSE). Cells were
immobilized with or without RDG-coupled alginate to
investigate the importance of the presence of cell
attachment peptides within the alginate. For some of the
cell lines, cell proliferation and multicellular spheroid
formation was independent of the presence of RGD. Cell
proliferation was measured by counting cells after degelling the foam using sodium citrate. Spheroids could
also be removed intact by de-gelling the alginate matrix
and further processed for histological staining. One
example shows the selective staining of apoptotic cells
within the spheroid. Use of alginate foams with
concomitant in situ immobilization of cells results in a 3D
cell culture model with the potential to approximate cell
proliferation and architecture within tissues or tumors.
The technology enables biomimetic approaches by
varying e.g. matrix elasticity, gelling ions, attachment
peptides and foam degradation making NovaMatrix-3D™
a versatile cell culture system. Portions of this work have
been funded with support from the METOXIA project no.
222741 under the 7th Research Framework Programme
of the European Union.
Keywords. 3D cell culture, alginate, multicellular
spheroid, scaffold
(17.P2) 3D PLLA SCAFFOLDS BY DIRECTIONAL
THERMALLY INDUCED PHASE SEPARATION (TIPS):
ARCHITECTURAL TUNING AND BIOLOGICAL VALIDATION
Mandoli C (1), Turella F (2), Forte G (1), Campana PT (3),
Traversa E (1)
1. National Institute for Materials Science (NIMS), Japan;
2. Dipartimento di Ingegneria Meccanica - Settore
Materiali, Universita' di Padova. Italy; 3. Universidade de
São Paulo, Escola de Artes, Ciências e Humanidades. Brazil
The role ascribed to the scaffold architectural
organization, as dictated by tissue engineering paradigms,
is foremost. As an artificial endoskeleton, the scaffold is
requested to provide optimal frameworks for the seeded
cells to organize into a functional tissue. Directional
thermally induced phase separation (dTIPS) is a versatile,
cost-effective technique for fabricating highly porous
scaffolds from different materials, having fully tailorable
porosity, and strongly anisotropic pore architectures.
Consequently, dTIPS scaffolds represent an ideal support
for the growth of biological tissues that exhibit gradient
morphology, such as bone, tendons, ligaments, nerves,
liver, pancreas, and in particular blood vessels. The
reconstruction of vascular grafts is in fact a prerequisite
when the growth of thick tissues is needed.
In the present work, we investigated the effect of the
process parameters, such as cooling temperature, (-30°C
≤ Tc ≤ +5°C), cooling time (2.5 h ≤ tc ≤ 32 h), and polymer
concentration (1.5 to 6.5 wt%), on the pore
microstructure of poly(L-lactic acid) 3-D scaffolds made by
dTIPS. The scaffolds exhibited highly ordered dendritic
domains, having overall porosities up to 95%, and
interconnectivity over 98%. By controlling the cooling
regime and polymer concentration we were able to tune
the pore diameter from few tenths of micrometers up to
260 μm, while keeping the peculiar pore hierarchy
unaltered, accompanied by a decrease in scaffold
compression modulus from 8 to 1 MPa. Moreover, the
biological validation assessed after 7 DIV of mesenchymal
stem cells culturing, evidenced massive scaffold
colonization.
In summary, the possibility to scale up and down the pore
architecture in dTIPS scaffold by one order of magnitude
by simple adjustments of the process parameters, may
allow creating a gradient porosity for in-growth of
complex tissues at any length scale (e.g., from macro to
micro blood vessels) in one single construct.
Keywords. PLLA scaffold, Thermally Induced Phase
Separation, mesenchymal stem cells, vascular tissue
(17.P3) INFLUENCE OF SCAFFOLD ANISOTROPY ON
TENOGENIC
MESENCHYMAL
STEM
CELL
DIFFERENTIATION - ALIGNED COLLAGEN I NANOFIBRE
SCAFFOLDS FOR POTENTIAL ROTATOR CUFF REPAIR
Rackwitz L (1), Hallinger R (1), Broermann R (1), Pullig O
(1), Rudert M (1), Nöth U (1)
1. Orthopaedic Center for Musculoskeletal Research, Dept.
Tissue Engineering/Regenerative Medicine, University of
Würzburg, Germany
Introduction. The utilisation of cell-seeded, biomimetic
scaffolds that reflect the high anisotropy (collagen I fibre
alignment) of native rotator cuff tissue might be
promising for the reconstruction of substantial defects of
the rotator cuff. Collagen I electrospinning was modified
using a rotating target to obtain nanofibre scaffolds (NFS)
with a different degree of anisotropy to investigate the
influence of fibre alignment on the behaviour of bone
marrow derived mesenchymal stem cells (MSC).
Material and Methods: Collagen I was isolated and
purified from rat-tail tendon. Collagen I was electrospun
onto a rotating mandrel at various speed (0,3 -10m/s).
The resulting NFS were characterised by scanning
electron microscopy (SEM) and mechanical testing.
Collagen I-NFS were seeded with bone marrow derived
MSCs (2 x 105/scaffold) and cultured under static
conditions in serum supplemented (10% FCS) medium for
up to 21 days. Cell orientation, tenogenic marker gene
expression (RT-PCR) and histological appearance were
evaluated at defined time points.
Results. Increased linear translation of the rotating
mandrel led to a higher fibre alignment (>90% at 10 m/s)
and increases tensile properties of the scaffolds. MSC
seeded on aligned NFS showed a high degree of cell axis
orientation parallel to the fibre alignment (>90% at 10
m/s) in contrast to a random orientation on non-aligned
NFS (0,3 m/s). RT-PCR revealed higher expression of
tenogenic marker genes (Scleraxis, Elastin, Col I) in
aligned vs. non-aligned scaffolds.
Conclusion. The consideration of ultrastructural aspects
of the target tissue is a crucial parameter in the process
of scaffold design for MSC-based tissue engineering
approaches. High anisotropy of collagen I-NFS supported
mechanical properties, MSC orientation and tenogenic
marker gene expression. Thus, underlining the superior
potential of aligned collagen I-NFS in MSC-based
approaches for the reconstruction of tenogenic tissues.
Keywords. Electrospinning, anisotropy, stem cell,
tenogenic differentiation, nanofiber
(17.P4) A COLLAGEN MATRIX ACTIVATES THE ERK
PATHWAY AND IMPROVES THE SURVIVAL AND
FUNCTION OF ENDOTHELIAL PROGENITOR CELLS
Marier J (1), Kuraitis D (1), Hou C (1), Zhang Y (1),
Vulesevic B (1), Ruel M (1), Suuronen EJ (1)
1. University of Ottawa Heart Institute, Department of
Cellular and Molecular Medicine. Canada
Introduction. Biomaterials are being developed to
augment the efficacy of endothelial progenitor cell (EPC)
therapy. EPC transplantation with a collagen matrix was
previously shown to be superior to EPCs alone for
restoring function to ischemic tissue. This study explored
a possible mechanism through which the matrix may
confer improved EPC therapy, specifically investigating
activation of the ERK pathway, which is involved in the
transduction of external signals to normalize intracellular
activities.
Methods. Human EPCs were cultured on fibronectin
(control) or a collagen/chondroitin sulfate-C matrix, crosslinked with glutaraldehyde. Cell lysates were probed for
ERK using Western blotting. Flow cytometry was
performed to assess cultures for progenitor cells (CD34,
CD133), for endothelial cells (CD31, CD144); and for
proliferation (EdU). Migration and adhesion of cells, with
or without ERK inhibitor (PD98059), were assessed.
Finally, cells were exposed to serum deprivation, and
viability was assessed using 7-AAD staining.
Results. Increased ERK1 (1.4-fold) and ERK2 (1.1-fold)
phosphorylation was observed in matrix-cultured cells
(p≤0.05), indicative of greater ERK activity. Proliferation
of CD133+ and CD133+CD34+ cells was increased on the
matrix compared to fibronectin (by 2.9- and 1.6-fold,
respectively; p≤0.02). Adhesion potential was greater on
collagen (4.0-fold; p=0.02), and 40% (p=0.02) more
matrix-cultured cells were observed to migrate. When
ERK inhibitor was applied, the differences between
treatments in adhesion and migration were abrogated.
After serum deprivation, there were 3.8-fold (p=0.07)
more viable CD34+ cells and 7.8-fold (p=0.02) more viable
CD133+ cells on collagen matrix.
Conclusion. A collagen matrix confers pro-survival and
proliferative signals for progenitor cells, and enhances cell
adhesion and migration capacity, mediated by the upregulation of ERK. The use of collagen matrices is
promising for enhancing cell-based regenerative
therapies.
Keywords. Endothelial Progenitor Cell; ERK; Cell culture;
Cell therapy
(17.P5) RECOMBINANT SPIDER SILK PROTEINS FOR
BIOMEDICAL APPLICATIONS
Hedhammar M (1), Widhe M (1), Jansson R (1), Johansson
U (1), Nordling K (1), Rising A (1), Johansson J (1)
1. Swedish University of Agricultural Sciences. Sweden
Spider silk is made up of unique proteins, spidroins, with
a tripartite composition; an N-terminal non-repetitive
domain, a highly repetitive central part composed of ~100
poly-Ala/Gly-rich co-segments, and a C-terminal nonrepetitive domain. Recent data on the N- and C-terminal
domains indicate that they have different specific
functions in the formation of spider silk fibres.
Miniaturized spidroins have been designed by combining
the terminal domains with a limited number of repetitive
segments
and
produced
recombinantly.
Such
miniaturized spidroins have been found to recapitulate
the properties of native spidroins to a surprisingly large
extent, provided that they are produced and isolated in a
manner that retain water solubility until fibre formation is
triggered. Moreover, recombinant spidroins can be
genetically modified to incorporate specific cell binding
motifs or improve mechanical strength. Herein, we
investigate some steps towards the realization of the
potential of recombinant spider silk for biomaterial
applications.
Miniature spidroins that include the C-terminal domain
can form macroscopic fibres within hours. When the Nterminal domain also is included, immediate selfassembly is observed at pH values below 6.4 (as observed
in the spinning duct of the spider), while the protein can
be stored for days in soluble form above pH 7 (as
observed in the gland of the spider). These properties can
be used in the development of a controlled
polymerization process. Generally, the self-assembly
process of these miniature spidroins seems robust, as
also modified variants, e.g. those with incorporated cell
binding motifs, can be processed into various formats,
such as free standing films, porous foams, capsules and
3D meshes. These results, together with the facts that the
silk matrices are of non-animal origin, mechanically
robust, easily sterilized, biodegradable and well tolerated
in vivo, hold promise not only for in vitro cell culturing,
but also for tissue engineering applications.
Keywords. Silk scaffold self-assembly biomimetic
(17.P6) A NOVEL DESIGN OF AN ARTIFICIAL ISLETCARRIER; THE EVALUATION OF ISLET SUPPORT,
ADHERENCE AND FUNCTION
Johansson U (1), Karin Åvall (2), Anna Rising (3), Ingrid
Schenning (3), Jan Johansson (1), Sewrgei Zaitsev (2), My
Hedhammar (1), PO Berggren (2)
1. Swedish University of Agricultural Sciences, Sweden;
2. Karolinska Institute, Sweden; 3. Spiber Technologies
AB,Swedish University of Agricultural Sciences, Sweden
Introduction. Transplantation of the islets of Langerhans
is one promising treatment for diabetes. Unfortunately
currently available procedures suffer from low efficacy
due to loss of function and survival of the pancreatic cells.
The low success rates are incompletely understood but
prior to transplantation, during islet isolation, the
environment surrounding the cells is disrupted. Therefore
establishment of an environment optimized for islets is
necessary for the design of a possible artificial, isletcarrier for transplantation. In order to do so, a highly
versatile biomaterial is needed as a scaffold.
Experimental methods. Recombinant spider silk, 4RepCT
is a strong and highly versatile material that can acquire
various forms e.g. three-dimensional fiber meshes, foams
or films1,2.
This newly generated synthetic variant of spider proteins;
both the wild type and variants modified by incorporation
of different intergrin and laminin related cell-binding
motifs (e.g RGD, IKVAV and YIGSR) was used to define an
environment for pancreatic islet adherence, islet function
and survival after isolation.
Isolated human and mouse pancreatic islets were
cultured up to 5 days either plated onto wells coated with
the 4RepCT protein in the various forms or without the
protein (control islets).
Results and discussion. The islet adherence to the
4RepCT various forms showed that both human and
mouse islets do adhere with an increased number to the
foam structure. There is also a preference for adherence
onto the foam with RGD cell-binding motif. The islets
plated on the 4RepCT were functionally active
demonstrating insulin release both under basal glucose
concentration and its’ stimulation with increase in
concentration of glucose.
Conclusion. The properties of 4RepCT can be used as
scaffolds mimicking the natural cell environment thus
providing support for the islets of Langerhans after
isolation.
Acknowledgements. The authors would like to thank
Vinnova and Barndiabetesfonden for providing financial
support to this project”.
Keywords. Recombinant spider silk, Islets of Langerhans
(17.P7) COMPOSITE SCAFFOLDS FOR VASCULAR TISSUE
ENGINEERING
Martorina F (1), Grandi C (1), Lora S (1), Dalzoppo D (1),
Panigotto PP (1)
1. Dept. Pharmaceutical Sciences, University of Padova,
Italy
Introduction. Extracellular matrix (ECM) influences
cellular response by interacting with cellular adhesion
molecules, growth regulators, binding proteins1,2,3. Our
idea is to realize scaffolds composed of synthetic polymer
integrated with lyophilized decellularized aortic matrix
(DAM) which should introduce specific attachment sites
for cell proliferation.
Methods. Bovine DAM was obtained with the detergentenzymatic method of Meezan4. A 1:1 (w/w) mixture of
DAM homogenate and polyvinyl alcohol (PVA) aqueous
solution was used to realize small-diameter vascular
scaffolds by low temperature treatments. After scaffolds
analysis by SEM, their citocompatibility was evaluated by
seeding endothelial cells. Cell presence was evaluated by
DAPI, H&E stainings and Movat's pentachrome technique.
DAM has been also analyzed by proteomic methods.
Results. Composite scaffolds were realized using a
mixture of PVA and lyophilized DAM. Proteomic analysis
evidenced ECM proteins like collagen I and VI. The threedimensional structure of the scaffolds has been evaluated
by SEM analysis. After in vitro seeding, with human
endothelial cells, scaffold sections have been stained with
DAPI and H&E to confirm the presence of cells and with
Movat’s pentachromic to stain typical ECM proteins. Cells
proliferated only on constructs conditioned with DAM
matrix, evidencing its specific role on cell attachment.
Conclusions. The presence of ECM regions in DAM
based/PVA scaffolds created specific attachments sites
for cell growth. Further analysis will be necessary to
evaluate the mechanical behaviour of the PVA scaffolds
after cell colonization. Once optimized all the conditions
for in vitro cell growth, we will try to implant in vivo these
vascular constructs.
References.
1. Friedl, P. et al. Microsc Res Tech, 43, 369, 1998
2. Badylak, S.F. et al. Acta Biomater, 5, 1, 2009
3. Grandi C. et al. 23rd ESB 11-15 Sept 2010, Tampere
(Finland)
4. Meezan, E. et al. Life Sci, 17, 1721, 1975
Keywords. Scaffolds, vascular graft, extracellular matrix,
tissue engineering
(17.P8) DEVELOPMENT OF BIOACTIVE PCL MATRICES
FOR TISSUE ENGINEERING OF LIGAMENT
Huot S (1), Rohman G (1), Migonney V (1)
1. Université Paris 13 Institut Galilée. France
Introduction. Ligament tissue engineering needs
appropriate source cells and growth matrix to support cell
proliferation and collagen synthesis. To control cell
response, new porous poly(ε-caprolactone) (PCL)
scaffolds were modified by grafting bioactive polymers,
poly(sodium styrene sulfonate) (PolyNass), that can
induce difference in fibroblast morphology and cell
activity during in vitro assay1. In the present work, cell
behaviour was first estimated onto 2D-PCL films and
thereafter into 3D cross-linked polymer scaffolds.
Methods. PCL films were manufactured by spin-coating.
Porous cross-linked PCL scaffolds were obtained using a
particulate-leaching process and paraffin beads as
porogen agents. For both films and scaffolds, surfaces
were functionalized through radical polymerization of
Poly(NaSS) after sample ozonation. Evidence of grafting
was provided by a toluidin blue colorimetric method and
X-ray photoelectron spectroscopy (Pr. David Castner,
NESAC/BIO, Seattle, USA). The porosimetry of porous
scaffold was analyzed by scanning electron microscopy.
Biological assays were carried out using McCoy cell line.
Discussion and Conclusion. Porosity in the range of 7580% was obtained for cross-linked PCL scaffolds in
agreement with the amount of porogen incorporated.
Spherical macropores were obtained with a remarkable
interconnection. Toluidine blue assay suggests an
homogeneous grafting of bioactive polymer on surface
samples. Cell response on grafted or non-grafted samples
indicates absence of toxicity. First results are encouraging
and further in vitro investigations have to be done.
References. 1. Ciobanu M. et al. Radical graft
polymerization of styrene sulfonate on poly(ethylene
terephthalate) films for ACL applications : « grafting from
» and chemical charactzerization. Biomacromolecules
2006; 7: 755-760
Keywords. Tissue engineering ligaments porous scaffolds
(17.P9) FABRICATION OF 3D CHITOSAN SCAFFOLDS
USING AN INVERSE PLOTTING METHOD
Lee H (1), Jeon HJ (1), Kim YB (1), Kim GH (1)
1. Chosun University, Republic of Korea
To create regenerated damaged tissues, cells are
attached and cultured onto a scaffold that is ultimately
implanted at the injured area of the functioning tissue, so
that the scaffold should be biocompatible and
biodegradable material. In two-dimensional scaffold, cells
are restricted to spread and attach to flat surface, so that
biophysical properties of the scaffold, which should
provide a spatial effect, may not be applied in the
implanted body. However, three-dimensional (3D)
scaffolds provide physical signals to guide cell
colonization as well as chemical signals of cell-binding
sites to support cell attachment and proliferation. To
achieve the ideal spatial architecture of the scaffold, solid
free-form fabrications (SFFs) have been introduced to
construct scaffolds in a layer-by-layer manner. Since the
SFFs can provide scaffolds with complex internal
structure, which cannot difficult with conventional
fabricating methods, the techniques are unique methods
for designing scaffolds. Generally, chitosan scaffolds have
been fabricated as porous structures by freeze-drying
process and electrospinning process. However, more
work for fabricating the chitosan scaffold should be
required due to difficult control of pore size and low pore
interconnectivity. To overcome these structural problems
of chitosan scaffold, we adapted a combined technology
of inverse plotting method with a sacrificing mold and
freeze-drying method. Using this method, we can acquire
a highly porous and stably pore-interconnected
structured 3D chitosan scaffold. To observe the feasibility
as a scaffold, we cultured MG63 cells in the scaffold and
the results were compared with a conventionally
designed spongy type scaffold.
Keywords. Chitosan, 3D scaffold, Bone
(17.P10) SCAFFOLDS TAILORED FOR BONE TISSUE
REGENERATION: EFFECT OF BIOCERAMIC FILLER
CONTENT ON ELECTROSPUN MEMBRANE PROPERTIES
Rajzer I (1), Chrzanowski W (2), Kwiatkowski R (1),
Menaszek E (3), Janicki (1)
1. Institute of Textile Engineering and Polymer Materials,
ATH University of Bielsko-Biala, Poland; 2. The Faculty of
Pharmacy, The University of Sydney, Australia; 3.
Departament of Cytobiology, Collegium Medicum, UJ
Jagiellonian University, Poland
Introduction. A large number of composite scaffolds have
been trailed for the tissue engineering applications,
however, a search for an optimal scaffold properties and
fabrication conditions is one of the key directions of
tissue engineering. The incorporation of nanofillers into
polymer matrix enhanced mechanical properties, and
improved osteoblast responses. More favourable cell
responses are typically associated with the chemistry,
topography and mechanical properties of the scaffolds,
which are tailored by inorganic fillers. Our aim was to
fabricate electrospun membranes modified with different
ceramic fillers and assess a function of the filler chemistry
on bioactivity. The scaffolds are intended for bone
applications.
Experimental methods. Membranes were electrospun
from polymer/ceramic solutions. Polymer matrix: PLDL
(PURAC), PCL (Sigma-Aldrich). Ceramic fillers: n-HAp
(AGH-Poland), TCP (Plasma-Biotal). The solutions were
spun at a working distance of 20cm, driving force of 30kV.
The solution flow rate was 15ml/h. The membranes
complex structure and their chemistry were characterised
using SEM, FTIR, and WAXD. The mechanical properties
were assessed on the basis of tensile tests. Biomimetic
growth of the apatite on the surface of biomaterials after
incubation in SBF was confirmed by SEM, EDX, WAXD and
FTIR.
Results and discussion. SEM after 7 days of incubation in
SBF revealed dense and uniform apatite layers, with
typical for apatite globular structure. Differences in ability
to apatite grains forming were observed between
samples with different fillers. The occurance of apatite
layer was detected in FTIR spectra after only 3 days of
incubation in SBF for PLDL/n-HAp samples, while for PLDL
samples a very weak FTIR bands associated with HAp
appeared after seven days.
Conclusion. These studies demonstrated that the
incorporation of ceramic filler into electrospun
membranes improved bioactivity, which was found to be
related to the chemistry of the filler.
Acknowledgments. Polish Ministry of Science and Higher
Education (project: N N507550938).
Keywords. Scaffolds, bone tissue, hydroxyapatite, TCP,
PLA, PCL
(17.P11) THE BIOMIMETIC POLYLACTIDE/ BETATRICALCIUM PHOSPHATE SCAFFOLD AS BONE GRAFT
FOR TISSUE ENGINEERING
Yen KC (1), Lin JH (2), Yao CH (3), Lin FH (1)
1. Institute of Biomedical Engineering, National Taiwan
University, Taiwan, R.O.C; 2. Institute of Textile
Engineering, Feng Chia University, Taiwan, R.O.C.; 3.
Department of Biomedical Imaging and Radiological
Science, Chia Medical University, Taiwan, R.O.C.
In this study, we took polylactide (PLA) filaments to form
3-dimensional braids by using 16-spindle braid machine
and enabling its structure to possess the even holes and
the tunnels, and then we stuffed the fabric into the betatricalcium phosphate (β-TCP) tube to adjust its
mechanical stress, which is similar to the human bone’s
structure. In vivo results indicate that polylactide/ betatricalcium phosphate scaffold can promote contact
osteogenesis.
Keywords. Polylactide, braid, tricalcium phosphate, bone
graft
(17.P12) INFLUENCE OF TCP CONTENT ON CHITOSAN
AGGLOMERATED SCAFFOLD PROPERTIES
Kucharska M (1), Walenko K (2), lewandowska-Szumieł M
(2), Brynk T (3), Ciach T (1)
1. Biomedical Engineering Laboratory, Faculty of Chemical
and Process Engineering, Warsaw University of
Technology; 2. Department of Biophysics and Human
Physiology, Medical University of Warsaw; 3. Faculty of
Materials Science and Engineering, Warsaw University of
Technology
Introduction. In hereby presented work a technique for
bone scaffold preparation is described. The method is
based on the agglomeration of chitosan/composite
microspheres. Authors present the fabrication process
and essential properties of the materials obtained.
Methods. In the first step microspheres (CH, CH_5%TCP
and CH_10%TCP) were extruded in the drop forming rate
from chitosan solution into precipitation bath. When
completely dried they were subjected to agglomeration in
presence of acetic acid, then subsequently neutralized,
washed and finally dried. The influence of TCP content on
physical and biological properties concerning HBDCs
culture was evaluated herein.
Results. The presented technique allows generating
porous materials with controllable shape, pore size
distribution and their interconnectivity. It was established
that microspheres extruded from CH solution only were
much smaller than those containing additionally TCP and
the diameters were enclosing in the range of 600 –
1000μm. As far as CH/TCP microgranules it was found
that the diameters were mostly over 1000μm (10001450). Young modulus established on the basis of stressstrain curves was similar for all of the materials and
equaled about 250 MPa. On the other hand we found
that compression strength decreased with increasing TCP
concentration. Our preliminary study concerning HBDC
culture did not show a clear influence of TCP
concentration on the viability of the cells, but XTT
measured after 48h revealed values of the viability
enclosing in the range of 60 and 85% when compared
with control sample.
Conclusions. In contrast to many methods for porous
materials manufacturing, the presented technique
permits to fabricate scaffolds with well-developed surface
for cell attachment. Mechanical properties were found to
be similar to natural bones. Satisfactory viability of HBDCs
after 48h of a direct contact with the investigated
material in culture is promising. Further detailed studies
on the interaction between the chitosan scaffolds and
cells are planned.
Keywords. Chitosan, agglomeration, in vitro
(17.P13) MECHANICAL STIMULATION OF FIBROBLASTS
IN MICRO-CHANNELED NANO-CELLULOSE SCAFFOLDS
ENHANCES PRODUCTION OF ORIENTED COLLAGEN
FIBERS
Martinez H (1), Brackmann C (2), Enejder A (2),
Gatenholm P (1)
1. Polymer Technology, Department of Chemical and
Biological Engineering, Chalmers University of
Technology,
Sweden;
2. Molecular
Microscopy,
Department of Chemical and Biological Engineering,
Chalmers University of Technology, Sweden
Introduction. Current meniscal repairing methods do not
repair all the meniscal tears, especially those occurring in
the avascular region. Even though meniscal
transplantation offers the best results for radial tears,
complex tears and degenerative tissue, current research
shows that degeneration of the articular cartilage still
occurs. Tissue engineering of fibrocartilage is a promising
solution to restore the function of the joints. Our aim is to
mimic the ultrastructure of fibrocartilage and implement
it in a nano-cellulose matrix. The strength of fibrocartilage
is attributed to the high content and alignment of
collagen fibers. Therefore, it is of extreme importance to
control the orientation of the cells and their extra-cellular
matrix.
Methods. In this study, we have developed a novel
scaffold concept based on nano-cellulose (NC) produced
by bacteria (Gluconacetobacter xylinus) and perforated
by micro-channels to mimic the ultrastructure of the
outer portion of the meniscus. The scaffolds with microchannels (~350 µm diameter) were prepared and seeded
with 3T6 fibroblasts. A compression bioreactor was
designed and constructed to evaluate the effects of
mechanical stimulation on collagen production. Dynamic
compression was applied to the NC scaffold/cell
constructs at a frequency of 0.1 Hz and compression
strain of 5%. A static culture was used as control. The
laser-based nonlinear microscopy techniques second
harmonic generation (SHG) and coherent anti-stokes
raman scattering (CARS) were used to visualize collagen
fibers and cell arrangement, respectively.
Results. Results from SHG, CARS and brightfield
microscopy showed that the micro-channels facilitate the
alignment of the cells and collagen fibers. Furthermore,
collagen production is enhanced by mechanical
stimulation.
Conclusions. These results show that it is possible to
engineer a composite biomaterial consisting of a nanocellulose matrix reinforced with oriented collagen fibers
and having potential to be used for development of a
knee meniscus implant.
Keywords. Nano-cellulose, micro-channels, mechanical
stimulation, collagen fibers
(17.P14) DEVELOPING A INJECTABLE BIOFUNCTIONAL,
BIOMIMETIC HYDROGEL SCAFFOLD FOR REGENERATIVE
MEDICINE APPLICATIONS
Seelbach R (1), Peroglio M (2), Fransen P (3), Royo M (3),
Mata A (4), Alini M (2), Eglin D (2)
1. AO Research Institute (ARI) Davos, Switzerland/
Nanotechnology Platform (PT), Parc Científic Barcelona
(PCB), Spain; 2. AO Research Institute (ARI) Davos,
Switzerland; 3. Institute for Research in Biomedicine (IRB),
PCB, Spain; 4. Plataformes Tecnològiques. Parc Científic
Barcelona (PCB), Spain
Multifunctional, biomimetic hydrogels presenting unique
physical and biochemical signals that enhance and
orchestrate a variety of biological processes during tissue
regeneration would potentially be a great material
platform for new therapies and in vitro studies.
Thus, bottom-up design scheme employing a nanofunctional platform based on multifunctional dendrimers
and a thermo-responsive hyaluronan hydrogel are
reported. The dendrimers are branched polyethylene
glycol nano-structures with five end-groups that can bear
azide functions and peptides to interact with alkyne
groups (e.g. Huisgen 1,3-dipolar cycloaddition (CuAAC))
and specific cell-surface receptors thereby inducing
desired cellular responses.[1] Hyaluronic acid (HA) is a
major component of the extracellular matrix in
connective tissues, synovial fluids, and as a provisional
matrix in developing organs. Furthermore, the thermoresponsive HA composition prepared by the CuAAC of
propargylamide substituted HA with azido functional
poly(N-isopropylacrylamide) has been recently described
and explored as a biodegradable scaffold in regenerative
medicine therapies.[2]
The project goal was to prepare dendrimers bearing RGDS
and azide functionalities that could be grafted via CuAAC
onto the thermo-responsive HA compositions.
Dendrimers with 4 RGDS peptide and 1 azide, 4 RDSG
scramble peptide and 1 azide were grafted at a peptide
concentration of 0.005mM/ml. After physicochemical
characterizations of the biomaterials, the behavior of
human mesenchymal stromal cells (hMSCs) seeded onto
the biomimetic gels were studied in vitro for 1 week.
Alamar Blue, Trypan Blue assays and histology was
performed. Preliminary data indicated that the
dendrimers were successfully grafted onto the HA (1H
NMR). Also, the gelling and mechanical properties of the
thermo-responsive HA compositions were influenced by
the presence of the hydrophilic dendrimers. No
significant differences were observed in the hMSC
viability seeded on the HA gel containing the RGDS and
scramble peptides dendrimers after 7 days, indicating
that both biological and mechanical cues are important
when developing a 3-D biomimetic matrix.
Keywords. Hydrogel, hyaluronan, dendrimer, peptide,
stem cells
(17.P15) TITANIUM SUBSTRATES COATED WITH
CALCIUM PHOSPHATE BY BIOMIMETIC METHOD
Rocha MN (1), Ribeiro AA (2), Andrade MC (3), Pereira LC
(1), Oliveira MV (2)
1. PEMM/COPPE/Federal University of Rio de Janeiro.
Brazil; 2. LATEP/DPCM/National Institute of Technology.
Brazil; 3. IPRJ/Rio de Janeiro State University. Brazil
Titanium (Ti) implants have been coated with calcium
phosphate (CaP) in order to improve their
osseointegration at the implant-bone interface, due to
the high biocompatibility of the mineral. This work aims
to study a biomimetic method for coating different Ti
substrates. It was used as substrates, micro (mTi) and
macroporous (MTi) titanium ASTM/grade 2 samples,
produced by powder metallurgy, with 19.56% and 61,38%
porosity, respectively, and commercially Ti ASTM/grade 2
dense sheet (CTi), with 2.8 µm medium roughness. The
samples were pre-treated for surface bioativation using a
1M NaOH solution followed by heat-treating at 200ºC in
air. Then they were immersed for 21 days in a simplified
solution (SS) at 37°C, based on CaCl2.2H2O and
Na2HPO4.2H2O salts. Phase characterization of the CaP
coatings was achieved by low angle X-ray diffractometry
(XRD) and Fourier transform infrared spectroscopy –
attenuated total reflectance (FTIR-ATR). The CaP
microstructure was identified by scanning electron
microscopy (SEM).
CaP precipitation with globular (MTi and CTi samples) or
plate-like (mTi sample) morphologies was observed by
SEM. The coatings XRD diffractograms showed different
CaP phases precipitated on the Ti substrates with
hydroxyapatite (HA) characteristic peaks for all samples,
octacalcium phosphate (OCP) for mTi sample and
carbonate apatite (CAp) for CTi and mTi samples. The
identified CaP phases were confirmed by FTI-ATR
analyses, which results were quite similar. Spectra from
mTi, MTi and CTi samples presented OCP and CAp
absorption bands. MTi and CTi samples also presented HA
absorption bands. The results demonstrated that the
biomimetic method used in this work successfully
precipitated bioactive CaP coatings onto the Ti samples
with different substrate types. However, adjustments in
the methodology will be necessary in order to obtain
continuous coating in shorter immersion times in SS.
Acknowledgements: CNPq, FAPERJ and Iberoamerican
Network BioFab-CYTED and LNLS/Campinas-SP/Brazil for
financial support.
Keywords. Calcium phosphate, Titanium, coating,
biomimetic
(17.P16) HYDROXYAPATITE COATING ON POLYMER
SCAFFOLD USING POLYDOPAMINE FOR BONE
REGENERATION APPLICATIONS
Yang HS (1), Park JY (2), La WG (2), Kim BS (2)
1. Department of Bioengineering, Hanyang University,
Seoul 133-791, Republic of Korea; 2. School of Chemical
and Biological Engineering, Seoul National University,
Seoul 151-744, Republic of Korea
Introduction. Biodegradable polymer/ceramic composite
scaffolds for bone regeneration applications are
advantageous over either biodegradable polymer or
ceramic alone. This study describes a simple and fast
method to coat polymer scaffolds with hydroxyapatite
(HA). Dopamine is a peptide sequence found in mussel
adhesive protein. It was investigated whether
polydopamine (DOPA)-coated polymer scaffolds can be
coated with HA nanoparticles.
Materials and Methods. Polyglycolic acid (PGA) meshes
were coated with HA by immersing the scaffolds in a 2-(Nmorpho1ino)ethanesulfonic
acid
buffer
solution
containing polydopamine (2 mg/ml) and HA nanoparticles
(20 and 5 mg/ml) for various periods of time. HA coating
on scaffolds were examined by selective staining of
ceramic particles, scanning electron microscopy,
attenuated total reflectance Fourier transformed-infrared
spectroscopy, X-ray photoelectron spectroscopy, and
energy-dispersive spectroscopy. To evaluate bone
formation efficacy of scaffolds in vivo, PGA scaffolds,
DOPA-coated PGA (DOPA-PGA) scaffolds, and HA/DOPAcoated PGA (HA-DOPA-PGA) scaffolds were implanted to
critical size defects in mouse skulls for 8 weeks.
Results. Various analyses showed that DOPA coating can
efficiently induce HA nanoparticle adsorption on PGA
mesh surfaces. Substantial HA coating on PGA scaffolds
was achieved within 24 hours of incubation. Soft X-ray
radiography,
microcomputed
tomography
and
histological analyses showed that bone regeneration in
vivo was more extensive on HA-DOPA-PGA scaffolds
compared to the other scaffolds.
Conclusion. DOPA offers an efficient and simple method
for HA coating on polymer scaffolds. HA-polymer
composite scaffolds fabricated with this method
exhibited enhanced bone formation efficacy as compared
to the polymer scaffolds.
Acknowledgement. This study was supported by a grand
(A101539) from the Korean Health 21 R&D Project,
ministry of Health and Welfare, Republic of Korea.
Keywords. Bone regeneration, hydroxyapatite composite,
polydopamine
(17.P17) SYNTHETIC MATRIX-MIMETIC POLYPEPTIDE
CONSTRUCTS
ENHANCE
ATTACHMENT
OF
MESENCHYMAL CELLS TO DIVERSE SCAFFOLD SURFACES
Szepesi Á (1), Szigeti A (1), Tátrai P (2), Szabó I (3), Mező G
(3), Német K (4)
1. Creative Cell Ltd., Budapest, Hungary; 2. Department of
Service, Budapest, Hungary; University of Debrecen,
Debrecen, Hungary; 3. Research Group of Peptide
Chemistry, Hungarian Academy of Sciences, Eötvös Loránd
University, Budapest, Hungary; 4. Department of
Service, Budapest, Hungary; Creative Cell Ltd., Budapest,
Hungary
For both bone tissue regeneration and implantation,
efficiency of cell attachment to the scaffold or implant
surface is critical to success. However, several widely
used surgical and implant materials have limited ability to
promote cell adhesion. Failure of cells, either engrafted or
host, to adhere to the surface may impede regeneration
or lead to implant loosening. In the present work, we
have tested the ability of synthetic polypeptide constructs
to improve attachment of various mesenchymal cells
(human mesenchymal stem cells [MSCs] isolated from
adipose tissue or differentiated from embryonic stem
cells, as well as MSC-like human foreskin fibroblasts) to
diverse surfaces such as non-tissue-culture plastic,
titanium, bone substitute of bovine origin (Bio-Oss®,
Geistlich Biomaterials), and a surgical mesh (TIGR™,
Novus Scientific). The polypeptide constructs consisted of
a polylysine backbone decorated with matrix-mimetic
oligopeptide motifs attached to spacer arms. Surfaces
were functionalized by simple physical adsorption of the
polypeptide conjugates. Attachment, survival, and
differentiation of cells was followed up to 8-21 days by
fluorescence and phase contrast microscopy, viability
assays, as well as fluorescent and Alizarin red staining.
Our observations confirmed that the polypeptide
conjugates increased the affinity of surfaces to cells with
efficiency comparable to that of fibronectin. As these
synthetic polypeptide conjugates can be manufactured in
a reproducible and cost-efficient manner, can be
lyophilized and stored indefinitely, are easily
reconstituted, and once applied to a surface remain inert
under normal conditions, they may provide a reasonable
alternative to recombinant protein-based surface
treatment. This work was supported by the Hungarian
National Office for Research and Technology (NKTH,
BIO_SURF).
Keywords. Extracellular matrix-mimetic peptide, bone
tissue regeneration, mesenchymal cell
(17.P18) GENERATION OF BIOARTIFICIAL HEART TISSUE
BY COMBINING 3D GEL BASED CONSTRUCT WITH
DECELLULARIZED MATRIX
Vukadinović Z (1), Dorfman SE (1), Horvath T (1),
Venturini L (2), Hilfiker-Kleiner D (3), Haverich A (4),
Hilfiker A (1)
1. Leibniz Research Laboratories for Biotechnology and
Artificial Organs (LEBAO), Hannover Medical School,
Hannover, Germany; 2. Department of Haematology,
Haemostaseology,
Oncology
and
Stem
Cell
Transplantation, Hannover Medical School, Hannover,
Germany; 3. Department of Cardiology and Angiology,
Hannover Medical School, Hannover, Germany; 4.
Department of Cardiac, Thoracic, Transplantation and
Vascular Surgery, Hannover Medical School, Hannover,
Germany
Introduction. A central problem in generating bioartificial cardiac constructs (BCC) in vitro is the efficient
supply of 3-dimensional tissues with nutrients and
oxygen. In order to create a functional, suturable implant,
we combined a gel based cardiac construct, with
decellularized porcine small intestinal submucosa (SIS)
and analyzed the reorganisation of seeded cells.
Methods. Isolated rat neonatal heart cells were mixed
with collagen I, and Matrigel and casted onto SIS, which
were pre-seeded with a monolayer of cells from the same
preparation either with or without supplementation of
7% GFP labelled rat endothelial (RHE) cells. During the
cultivation period (14 d) BCC were functionally
investigated in respect to frequency and direction of
contractions. Histological and immunohistological stains
were conducted to observe cellular organisation of BCCs.
Results. All BCC contracted spontaneously and
rhythmically, as one unit, in the direction of collagen
fibres within the SIS, with an average rate of 200 beats
per minute. Cells within the constructs appeared in
aligned manner, and cardiomyocytes were elongated and
well organized. A dense CD31 positive, 3D network of
endothelial cells through the whole construct could be
observed after 7 days. GFP labelled RHE cells were found
not only along the monolayer between SIS and the gel
construct, but also upwards growing through the gel
construct up to the top, and also downwards into the SIS.
Moreover, after 14 days, pre-existing decellularized vessel
structures of the SIS were re-populated to a high degree
(Fig 1).
Conclusion. A 3D tubular-like network built by endothelial
cells, being a cellular component of neonatal rat heart
isolates in a solid bio-artificial cardiac construct, may offer
a connecting system for the vascularization of this tissue
upon implantation. Thus, it might be an important
precondition for the survival of thicker myocardial
replacement constructs.
Keywords. Small-intestinal submucosa; bio-artificial
cardiac construct; vascularization; endothelial cells
Re-populated vessel structures of the SIS with stably
transfected GFP-tagged RHE cells following 14 days of
cultivation
(17.P19) PERFUSION-BASED 3D MICROTUMOR CULTURE
PLATFORM FOR CANCER CELL CULTURE AND ANTICANCER DRUG TESTING
Liu XH (1), Cui ZF (1)
1. Institute of Biomedical Engineering, Department of
Engineering Science, Oxford University. UK
Many cell lines have been successfully cultured in
different three dimensional models in vitro. But 3D
culture has the limitation of nutrition and oxygenation
perfusion, which cannot be achieved by simple diffusion.
3D dynamic culture model, as a simulation of vascular
system, can significantly improve the cell viability in vivo.
In this study, a multiple parallel perfusion-based
bioreactor (TissueFlex®) was employed to study the
differences between static culture and perfusion culture
on cell activities and drug responds. Two commercial
available anti-cancer drugs (Paclitaxel and Cisplatin) were
tested on DLD1 and NCI/ADR cell lines in a monolayer and
three dimensional formats. Perfusion culture system is
believed to provide stable and physiological environment
by continually supplying culture medium and removing
waste medium. Cells show higher growth rate and higher
cell activity in perfusion culture than static. And for drug
treatments, cells shows significant different toxic
responds under perfusion and static culture for
monolayer and 3D culture. Cells cultured in perfusion
system are more sensitive to drug dose-response and
show lower growth inhibition, which indicates the
importance of providing suitable system to testing cellular
responds to drugs.
Keywords. Cancer cells; 3D culture; perfusion culture;
toxicity testing
Acknowledgements. Supported by the Acad. Sci. CR
(grants No. KAN400480701, IAAX00100902), and the
Grant Agency of the CR (grant No. P108/11/0794, LG
06063).
Keywords. Carbon nanoparticles, nanotechnology,
electrical conductivity, bone tissue engineering
(17.P20) HUMAN OSTEOBLAST-LIKE CELLS ON BORONDOPED NANOCRYSTALLINE DIAMOND THIN FILMS
Burdikova Z (1), Grausova L (1), Bacakova L (1), Kromka A
(2), Rezek B (2), Haenen K (3)
1. Institute of Physiology, Academy of Sciences of the
Czech Republic, Videnska 1083, CZ- 14220 Prague 4, Czech
Republic; 2. Institute of Physics, Academy of Sciences of
the Czech Republic, Cukrovarnicka 10, CZ- 16253 Prague
6, Czech Republic; 3. Institute for Materials Research
(IMO), Hasselt University & Division IMOMEC, IMEC vzw,
B-3590 Diepenbeek, Belgium
Introduction. Nanocrystalline diamond (NCD) is a
promising material for various biotechnologies, including
construction of biosensors, detection, separation and
purification of biomolecules, and surface coating of bone
implants. NCD films can be rendered to be electrically
conductive by doping with boron, which may increase
their attractiveness for cell colonization.
Methods. Nanocrystalline diamond (NCD) films were
deposited on silicon substrates by a microwave plasmaenhanced CVD process and doped with 133, 1000 and
6700 ppm of boron in the gas phase. The films were
seeded with human osteoblast-like MG 63 cells and their
adhesion, growth and osteogenic differentiation were
investigated. The adsorption of collagen I, an important
component of bone extracellular matrix, was also studied
by confocal laser scanning microscopy, two photon
microscopy and second harmonic generation microscopy
imaging.
Results. The electrical resistivity of the films decreased
from >10 MΩ (non-doped films) to 55, 0.6, and 0.3 kΩ
(doped films with 133, 1000 and 6700 ppm of B,
respectively). The increase in the number of MG 63 cells
in 7-day-old cultures on NCD films was most apparent on
NCD doped with 133 and 1000 ppm of B (152,500 ±
13,900 and 152,200 ± 10,400 cells/cm2, respectively,
compared to 112,900 ± 9,700 cells/cm2 on non-doped
NCD films). On NCD films with 6700 ppm of B, the cells
contained the highest concentration of focal adhesion
protein vinculin, measured per mg of protein. Similarly
the concentration of osteocalcin, an important marker of
osteogenic cell differentiation, increased with increasing
level of B doping. Boron doping also positively influenced
adsorption of collagen I and its production by cells.
Conclusions. Our results suggest that the potential of
NCD films for bone tissue regeneration can be further
enhanced by boron-doping.
(17.P21)
TRANSFERASE-CATALYZED
BIOMIMETIC
HYDROGELS FOR TISSUE ENGINEERING
Mosiewicz KA (1), Ranga A (1), Johnsson K (1), Lutolf MP
(1)
1. Institute of Bioengineering, Ecole Polytechnique
Fédérale de Lausanne (EPFL), Switzerland
Synthetic hydrogels are key elements in emerging
strategies for tissue engineering and cell biology.
However, the current shortage of highly specific and
biocompatible methods to form and functionalize these
materials hampers their wider use in pharmaceutical and
medical applications. To this end, we envision that
enzymatic cross-linking schemes could be an essential
and still underexplored option for biomaterials
development.
We present an application of phosphopantetheinyl
transferase (PPTase) for covalent cross-linking of
poly(ethylene glycol) (PEG)- based hydrogels. PPTase is an
enzyme that plays a key role in the biosynthesis of many
natural products and has been employed as a
biotechnological
tool
for
site-specific
protein
modification. PPTase performs a highly specific transfer of
phosphopantethyinyl residue of Coenzyme A (CoA) into
the active site of specific carrier proteins (CPs). In our
recently developed PPTase-based hydrogel system, crosslinking occurs between CoA-functionalized multi-arm PEG
1
macromer and a genetically engineered CP dimer.
Importantly, here we have explored the possibility of
replacing CP by a small synthetic peptide analog.
Chemically synthesized short CP was tested as a dimer or
was conjugated to multi-arm PEG, which offers the
opportunity for further optimization and modulation of
gel network architecture. In this study, the
physiochemical properties of hydrogels produced by
these different approaches are compared. Furthermore,
we show that using this enzymatic scheme, site-specific
modification of PPTase-hydrogels is possible, as
demonstrated by covalent incorporation of integrinbinding cell adhesion ligand in 2D as well as 3D cellular
assays.
In conclusion, PPTase-based hydrogels represent a novel
class of functional and bioactive materials which offer the
possibility of tuning physiochemical properties through a
rapid, highly specific cell-friendly cross-linking reaction.
Furthermore, the completely synthetic design of this
material is a key feature which may be relevant in clinical
settings. Consequently, we envision a wealth of useful
applications of this new gel system in cell biology and
tissue engineering.
Reference. 1. Mosiewicz, K. A.; Johnsson, K.; Lutolf, M. P.,
Journal of the American Chemical Society 2010, 132, (17),
5972.
18. ESB - TERMIS SYMPOSIUM:
BIOMECHANICS IN TISSUE
ENGINEERING
Chair: Damien Lacroix
Co-chair: Dominique Pioletti
Keynote speaker: Manuela Teresa Raimondi
Organizer: European Society of Biomechanics
Synopsis: Biomechanics plays a major role in the
development of tissue engineering approaches as it has
been recognized that mechanical stimuli acting directly
on cells affect gene expression. Therefore, throughout
the development of tissue engineering as a discipline of
bioengineering, the progress made in the design and
construction of bioreactors, and the morewidespread use
of bioreactors have allowed to understand better the
interactions between biomaterial scaffolds, cells and
mechanical stimuli, and have allowed to develop more
functional scaffolds for different applications of
regenerative medicine. More recently, progress has been
made in the development of in silico techniques that
enable to simulate the different biological processes
occurring in tissue engineering such as cell seeding, cell
proliferation and cell differentiation. These techniques
not only bring a better understanding in the
mechanobiological
processes
underlying
tissue
engineering but also provide tools to optimize the
bioreactor conditions for the development of functional
scaffolds and therefore avoid the experimental ‘trial and
error’ approach.
In this symposium new advances in the biomechanics of
tissue engineering will be presented. Contents of the
presentations in this symposium with a focus on
mechanical stimuli can include:
- Design of new bioreactors
- Mechanical loading on scaffolds
- Effect of mechanical stimuli in scaffolds in bioreactors
- In vivo mechanical stimulation of tissue regeneration
- Simulation of nutrient transport in bioreactors
- Simulation of mechanical stimuli in bioreactors
- Simulation of in vivo tissue growth and regeneration
(18.KP) MECHANOBIOLOGY OF CARTILAGE TISSUE
ENGINEERING
Raimondi MT (1)
1. Politecnico di Milano, Italy
To engineer a cartilaginous tissue in vitro, the basic idea is
to expand a cell population, to seed the cells on a
biomaterial, and to culture the construct until its
maturation into a functional tissue. An essential step
toward the obtainment of functional cartilage is to
control its growth process. This process depends on
various space- and time-varying biophysical variables of
the cell environment, primarily mass transport variables
and mechanical variables, all involved in the cell’s
biological response.
In the general aim to obtain a quantitative law for tissue
growth, in function of the above mentioned variables, we
have developed several growth models, in which the
cellular constructs are subjected to a flow of culture
medium and/or to cyclic pressurization on a macroscopic
scale, and computational modelling is used to quantify
variables of the biophysical field induced on the cells on a
microscopic scale.
Using this technique, we have quantified specific aspects
allowing to control the culture conditions. For perfusion
alone, we estimated the relationship between the global
production of matrix proteins by the cells, and the level of
fluid-induced shear exerted on the cells. For perfusion
combined to cyclic pressurization, we estimated the
relationship between the local level of oxygen tension
sensed by the cells, and the local up-regulation of hyaline
matrix protein production, in response to pressurization.
Our recent developments include a more advanced
growth model, featuring a mini-bioreactor system,
allowing local and non-destructive assays on the cellular
constructs, to be interfaced to a multiphysic model of
tissue growth, in which the known dependences are nonlinearly coupled.
Acknowledgements. This research is funded by the
grants: ‘Biosensors and Artificial Bio-systems’- Italian
Institute of Technology (IIT-Genoa); ‘5x1000-2009-HMED:
Computational Models for Heterogeneous Media’Politecnico di Milano; ‘3D Microstructuring and
Functionalization of Polymeric Materials for Scaffolds in
Regenerative Medicine’- Cariplo Foundation (Milano).
Keywords. Biomechanics, mechanobiology, regeneration,
model
(18.O1) BIOMECHANICAL CONCEPTS TO DESIGN
PERFUSION BIOREACTOR FOR ENGINEERING BONE
David B (1), Deschepper M (2), Petite H (2), Oddou C (3)
1. Laboratoire Mécanique des Sols, Structures et
Matériaux (MSSMat), UMR CNRS 8579, École Centrale
Paris, France; 2. Laboratoire de Bioingénierie et
Biomécanique Ostéoarticulaire (B2OA), UMR CNRS 7052,
Université Paris 7. France; 3. Laboratoire Matière et
Systèmes Complexes (MSC), UMR CNRS 7057, Université
Paris 7, France
One challenging task in engineering bone tissue with
bioreactor is to maintain an adequate balance between
high supply of medium and sufficiently low fluid shear
stresses applied to cells. This trade-off can be achieved in
designing a system based on the concepts in fluid
dynamics of porous media. Therefore, we designed a new
perfusion bioreactor, for the culture of bone constructs of
clinically-relevant size, using flow in fluidized bed [1].
Natural coral, a microporous and biocompatible material,
was used as three-dimensional scaffolds. This bioreactor
provided a stable environment of the cells in terms of
mechanical and physicochemical properties. The chamber
contains around 150 constructs (cell-seeded in cubic
samples of 9 mm3 volume) imbedded in the flowing cell
culture medium. Such constructs are settled with
randomized localization and orientation leading to a
complex design of the scaffold structure. The overall
substrate contained in the perfusion bioreactor can then
be roughly considered as a porous medium presenting a
large spectrum of m to 1 mm and an overall porosity
greaterµpore dimensions, from 100 than 50 %.
Accounting for the value of the applied perfusion mean
m/s) and the architectural characteristics of theµvelocity
(about 102 substrate, an approached evaluation of the
applied shear stress would be around 1 mPa. These
values are commonly advanced in case of noticeable
mechanotransduction effects of cells embedded within
three-dimensional substrates without risk of cell
detachment. Bone constructs engineered in this system
resulted in significantly high cell proliferation and
homogenous cell distribution. Furthermore, these bone
constructs were shown to be osteogenic when
transplanted subcutaneously in sheep. This techniques
thus appears to be particularly relevant to the production
of bioengineered bone with clinically-relevant volume.
[1] B. David & al., A perfusion bioreactor for engineering
bone constructs: An in vitro and in vivo study, Tissue
Engineering C (2010)
Keywords. Perfusion Bioreactor, Coral Scaffold, In Vitro
Study
(18.O2) THE INFLUENCE OF HYDROSTATIC PRESSURE ON
THE CHONDROGENESIS OF MESENCHYMAL STEM CELLS
EMBEDDED IN EITHER AGAROSE OR FIBRIN HYDROGELS
Steward AJ (1,2), Thorpe SD (1), Vinardell T (1), Buckley CT
(1), Wagner DR (2), Kelly DJ (1)
1. Trinity College Dublin, Ireland; 2. University of Notre
Dame, Indiana, USA
Introduction. Mechanical loads have been shown to play
an important role in the differentiation of mesenchymal
stem cells (MSCs). Hydrostatic pressure (HP) specifically
has been shown to affect extracellular matrix (ECM)
synthesis in vitro. Cell attachment is directly affected by
the scaffold substrate and plays a key role in
differentiation. The objective of this study was to
examine the interplay of cell attachment and hydrostatic
pressure on the chondrogenesis of MSCs.
Methods. MSCs were harvested from porcine bone
marrow and seeded into hydrogels that either permitted
(fibrin) or prevented (agarose) cellular attachment. The
hydrogels were subjected to 10 MPa of hydrostatic
pressure for 4 h/d at a frequency of 1 Hz for 5 days per
week. Scaffolds were cultured in a chemically defined
chondrogenic media, and cultured with different
concentrations of human TGF-β3. Samples were
biochemically analyzed and observed with confocal
microscopy.
Results. Confocal microscopy demonstrated that cells
seeded in fibrin attained a spread, flattened morphology,
while cells in agarose retained a round, spherical
morphology (Fig. 1A). Fibrin hydrogels permitted MSC
proliferation, while cell death occurred in the agarose
hydrogels. HP significantly decreased the proliferation of
MSCs in fibrin cultured in 1 ng/ml TGF-β3 (Fig. 1B).
Collagen accumulation was greater in fibrin hydrogels
subjected to HP in 10 ng/ml TGF-β3 (Fig. 1B). HP had no
influence on matrix accumulation in agarose hydrogels.
Conclusions. This study demonstrated that HP effects
cellular proliferation and matrix accumulation in fibrin
hydrogels, but has no effect on proliferation in agarose
constructs. Fibrin better supported cell viability and
accumulation of collagen relative to agarose. These
results demonstrate that cell-matrix interactions regulate
MSC response to HP.
Acknowledgements. Funded by a Naughton Fellowship
and SFI PIYRA [SFI/08/YI5/B1336].
Keywords. Hydrostatic Pressure, Cell-Matrix Interactions,
Mesenchymal Stem Cells
(18.O3) A NOVEL BIOREACTOR FOR THE SYSTEMATIC
DEVELOPMENT OF FUNCTIONAL 3D SCAFFOLDS FOR IN
SITU CARDIOVASCULAR TISSUE ENGINEERING
Smits AIPM (1), Driessen-Mol A (1), Bouten CVC (1),
Baaijens FPT (1)
1. Eindhoven University of Technology, Netherlands
Introduction. State-of-the-art cardiovascular tissue
engineering (TE) strategies are increasingly directed
towards an in situ TE approach. This approach is based on
using unseeded, ‘smart’ instructive scaffolds as
replacement grafts, promoting endogenous cell
recruitment and subsequent remodeling [1]. Clearly, the
interactions between the scaffold and circulating cells
under physiologic hemodynamic conditions play a pivotal
role in this process and determine the optimal scaffold
design. The aim of the current study is to develop an in
vitro model system for the systematic development of
such functional 3D scaffolds.
Methods. The model system consists of a custom-made
cross-flow chamber (CfC) that houses 3D scaffolds (Fig.
1A). The CfC is incorporated into a flow setup designed to
drive a cell suspension along the scaffold with physiologic
wall shear stresses (0.1-8 Nm-2) and perfusion pressures
(80-100 mmHg). Performance of the CfC was assessed
with computational fluid dynamics and validated
experimentally with fluorescent microbead (Ø10 µm)
tracing studies. For proof-of-principle, human peripheral
blood mononuclear cells (hPBMC) were isolated and
labeled with Cell Tracker Green (CTG). The hPBMC were
driven along a 3D electrospun scaffold under
physiological flow conditions and infiltration of CTGlabeled cells into the scaffold was analyzed.
Results. Computational predictions demonstrate a fully
developed flow in the region of interest, with a
homogenous wall shear stress distribution (Fig. 1B,C).
Consistently, microbeads followed a straight trajectory
without turbulations (Fig. 1D). Furthermore, achievable
levels of shear stress and perfusion pressure are within
the physiological range and are independently
controllable. Additionally, hPBMC infiltration and
adhesion could be monitored in real-time with confocal
microscopy during the cell studies. Studies on the effect
of scaffold architecture on cell recruitment under
physiologic hemodynamic conditions are ongoing.
Conclusion. Our model system provides an ideal
screening platform for the development and systematic
evaluation of functional 3D scaffolds for in situ
cardiovascular TE.
References. [1] A. Mol, A.I.P.M. Smits, C.V.C. Bouten and
F.P.T. Baaijens “Tissue engineering of heart valves:
advances and current challenges”, Expert Rev. Med.
Devices, Vol. 6, pp. 259-275, (2009).
Keywords. Cell-scaffold interaction hemodynamics
bioreactor
(18.O4) EARLY STAGE rMSC DIFFERENTIATION CAN BE
INDUCED BY FLUID FLOW IN THE ABSENCE OF
OSTEOGENICALLY SUPPLEMENTED MEDIA
McCoy RJ (1), Duffy G (1), O'Brien FJ (1)
1. Royal College of Surgeons in Ireland
Previous work in our laboratory has shown that
mechanical stimuli (fluid flow) can regulate osteoblast
osteogenesis when cultured on collagen-based scaffolds
in the presence of osteogenic supplements [1],[2],[3],[4].
The current focus of our work aims to determine if
mesenchymal stem cell (MSC) differentiation towards an
osteogenic lineage is similarly regulated by flow.
Furthermore, through de-coupling the effects of physical
and chemical stimuli, we seek to provide furthered
understanding regarding the individual contributions of
mechanically and chemically activated signalling
pathways on rMSC gene expression during initial stages of
differentiation. Rat MSC cell-seeded collagen-GAG
scaffolds, cultured in growth medium (no osteogenic
supplements), were exposed to oscillatory or steady flow
regimes for 49hr and compared to static controls.
Flow significantly decreased levels of SOX9 and
PPARgamma gene expression, transcription factors
associated
with
chondrogenic
and
adipogenic
differentiation, whilst maintaining levels of RunX2 (proosteogenic). Alkaline phosphatase (ALP) and integrin
alpha 1 (ITGA1) gene expression were down-regulated,
whilst osteopontin (OPN) and collagen type-1-alpha1(Col1A1) levels were maintained (Figure 1). Down
regulation of ALP and ITGA1 suggests cells are exiting a
proliferative state and entering differentiation, which is
supported by the transcription factor gene expression
data, with cells appearing to commit to an osteogenic
lineage. Changes in gene expression levels of later stage
osteogenic related proteins were not observed at this
early stage of differentiation.
This work highlights flow can play a significant role in
directing early stage rMSC osteogenic differentiation.
Further investigation is required to determine if flow
alone can direct cells into a mature lineage phenotype.
Ongoing studies culturing rMSC in the presence of
osteogenic supplements will endeavour to prize apart the
roles of chemical and mechanical stimuli on gene
expression during rMSC differentiation.
References.
[1] Jaasma et al. J. Biotech. 133:490-496, 2008.
[2] Jaasma and O'Brien. Tissue Eng Part A. 14:1213-1223,
2008.
[3] Partap et al. J Mater Sci Mater Med. 21:2325-30, 2008
[4] Plunkett et al. Tissue Eng Part A. 16:943-951, 2010.
Keywords. Mesenchymal Stem Cell; Perfusion Bioreactor;
Osteogenic Gene Expression; Collagen-based scaffolds
(18.O5) THE EFFECTS OF FLOW-PERFUSION ON
HYPERTROPHIC DIFFERENTIATION OF ENDOCHONDRAL
BONE CONSTRUCTS
Gawlitta D (1), Van Rijen MHP (1), Malda J (1), Dhert WJA
(1,2)
1. Department of Orthopaedics, University Medical Center
Utrecht, The Netherlands; 2. Faculty of Veterinary
Medicine, Utrecht University, The Netherlands
Endochondral bone tissue engineering is an attractive
strategy to circumvent vascularization issues as it involves
a cartilaginous transition tissue that is naturally avascular.
Attractive cells for this purpose are the bone marrowderived multipotent stromal cells (MSCs). There are
strong indications that mechanical cues can control
cellular differentiation. In particular, mild shear stresses
were shown to enhance hypertrophy in e.g. chondrocytes
(Wong et al., Bone 33, 2003). Therefore, we hypothesized
that the endochondral process of chondrogenic MSCs can
be enhanced by imposing appropriate mechanical cues,
such as flow-perfusion-induced shear stress.
Human MSCs were isolated, expanded and centrifuged to
form spherical aggregates. These were subsequently
mounted into 3D-printed porous, polycaprolactone (PCL)
scaffolds in basic chondrogenic differentiation medium
(Figure). The hybrid constructs were then allowed to form
a cartilaginous matrix for 18 days, before they were
transferred to a custom-built flow-perfusion system.
Controls were maintained under static culture conditions.
After 3 days, samples were harvested for RT-PCR of
chondrogenic marker genes, COL2A1 and SOX9 and
hypertrophic markers COL10A1 and BGLAP (osteocalcin).
Additionally, samples from both groups were processed
for histology and Western blot analysis of collagen type X
at day 28. Deposition of proteoglycans (Figure; in red) and
collagen type II are indicative of the formation of a
cartilaginous matrix, while collagen type X and matrix
mineralization indicate hypertrophic differentiation of the
newly formed tissue.
Feasibility of this novel hybrid construct assembly method
from cell aggregates and printed polymeric scaffolds was
shown. Additionally, the progression of differentiation of
the MSCs following mild shear stress stimulation in these
hybrid constructs can be evidenced by detection of
chondrogenic and hypertrophic markers. Demonstrating
that mechanical stimulation affects hypertrophic
differentiation is relevant for both the maintenance of
engineered cartilage and for inducing endochondral bone
formation.
Keywords. Mechanical, shear, MSC, hypertrophy,
endochondral
(18.O6) DAMPING PROPERTIES OF THE NUCLEUS
PULPOSUS
Pioletti D (1), Vogel A (1)
1. Laboratory of Biomechanical Orthopedics-EPFL,
Netherlands
Questions persist in the investigation of the viscoelastic
behavior of the nucleus pulposus (NP) of the
intervertebral disc. In particular, the damping properties
of the NP under physiological large deformations are still
to be addressed. Bovine coccygeal NP tissues have been
harvested and encapuslated into a deformable and
permeable device. The encapsulation device is composed
of a medical grade 40 μm pore size sintered steel filter, an
nonporous rigid disc, and a 100 μm thin
polydimethylsiloxane. The proposed approach allowed us
to monitor the water content of the samples during
mechanical tests which of primary importance in the
dissipation evaluation process (Figure 1). The specific
damping capacity of the NP in large compressive
deformations (12.5%) and for frequencies ranging
between 0.01 and 10 [Hz] was assessed using a paired
statistical study. Damping ranged between 18 and 33%
with a minima at 0.1 [Hz]. Because the NP can show both
fluid and solid behaviors, the specific damping capacity
used here was defined by dividing the energy loss
(hysteresis) by the work input. It represents the
proportion of energy that is dissipated into heat. This
energetic approach is particularly convenient to study
nonlinear viscoelastic materials such as biologic soft
tissues. In summary, in the present study, we introduce a
reliable method to address the damping properties of
hydrogels under large and physiological deformations,
and to investigate the damping properties of the
coccygeal bovine nucleus pulposus in order to provide
data for the design of nucleus replacement devices.
Keywords. Soft tissue, dissipation, nucleus pulposus,
hydrogel
(18.O7) INVESTIGATING THE POTENTIAL OF HIGH
FREQUENCY LOW MAGNITUDE (HFLM) LOADING
INTERVENTIONS FOR TENDON REPAIR USING A NOVEL
IN-VITRO LOADING SYSTEM
Adekanmbi I (1), Baboldashti NZ (1), Franklin S (1), Hulley
P (1), Poulsen R (1), Thompson M (1)
1. University of Oxford, United Kingdom
Introduction. Mechanical stimulation has been
postulated as an essential factor in maintaining tendon
health, and there are indications it may be beneficial for
promoting tendon repair. Several in-vitro studies have
examined the effects of mechanical stress on healthy
tendons by using loading frequencies of 0.01-3Hz since
such loading frequencies may occur during physical
exercise. More recently, studies have shown evidence for
the special effects of using high frequency low magnitude
(HFLM), loading regimes in promoting bone health and
counteracting bone disease. In this study, a novel in-vitro
loading system (IVLS) has been developed with the aim of
investigating the potential of HFLM stimulation for
tendon repair.
Materials and Methods. Tendon fascicles from male
Sprague Dawley rat tails (4-6months) were cultured in
centrifuge tubes (DMEM 10%FCS). Fascicles were
incubated under conditions of no load, static load, or
cyclic load, using a custom IVLS . Cyclic loading of
specimens was achieved using a pulsed electromagnetic
field to perturb a magnet suspended from the fascicle.
The load magnitude and frequency applied onto fascicles
was measured using a sensitive load cell and software
analysis. Live-dead staining was used to examine tissue
viability after 0 (fresh), 1, 4, and 7 days. Tensile testing
and a Glycosaminoglycan assay were performed to
measure biomechanical and extracellular matrix
alterations.
Results and Discussion. Preliminary results revealed that
the developed IVLS can sustain tissue viability for a
minimum of 7days subsequent to static and HFLM loading
interventions. The load frequency applied was confirmed
to be 20Hz and peak loads varied between 0.15-0.25N.
Furthermore, by day 4, fascicles cultured under static load
showed
significantly
higher
Modulus
and
Glycosaminoglycan content(figure 1)compared with load
deprived specimens (2 fold difference). These results
demonstrate the capability of the developed system for
investigating the potential of HFLM loading interventions
in promoting tendon repair.
Keywords. Tendon, mechanobiology, in-vitro loading
system, repair
Figure 1: The Glycosaminoglycan content for fresh tendon
fascicles, and cultured tendon fascicles after 4days of
unloading, and static loading
(18.O8) STRAIN INDUCED REMODELLING OF POTENTIAL
SCAFFOLDS FOR TISSUE ENGINEERED BLOOD VESSELS
Campbell EM (1), Mackle JN (1), Gatenholm P (2), Lally C
(1)
1. School of Mechanical & Manufacturing Engineering,
Dublin City University, Dublin, Ireland; 2. Department of
Chemical & Biological Engineering, Chalmers University of
Technology, Sweden
Introduction. The advantages of applying dynamic culture
conditions to cell-seeded scaffolds are well established in
terms of tissue maturation, extracellular matrix
formation, and enhanced mechanical properties [1].
Bacterial cellulose (BC) has been investigated as a
potential tissue engineered blood vessel scaffold [2]. This
study determines the biological response of cell-seeded
BC to dynamic culture conditions with the aim to extend
the study to repopulated decellularised porcine coronary
artery (PCA).
Methods. 15x15mm sections of BC were adhered to
Bioflex® culture plate membranes. Bovine aortic smooth
muscle cells (BASMC) were statically cultured on the BC
sections
at
a
concentration
of
300,000
cells/cm2. Following 72 hours of culture a mean cyclic
uniaxial strain of 6.5% with 3% amplitude was applied by
a Flexercell® FX-4000TM for 120 hours in humidified air
with 5% CO2 at 37°C. Cell infiltration was determined by
hematoxylin and eosin (H&E) staining and smooth muscle
α-actin was used to examine cell phenotype.
Results. H&E staining of cyclically strained BC showed
BASMC infiltration of 34%, which shows enhanced
infiltration compared to static controls (10%), see Fig.1.
The cells maintained their smooth muscle phenotype.
Conclusions. Following the successful remodelling of cellseeded BC under cyclic strain this study can be extended
to other scaffolds. Decellularised PCA has the capability
for cell attachment as demonstrated by static culture of
BASMC [3]. Cell culture experiments will be performed to
characterise the proliferation, migration and infiltration
of repopulated cells in decellularised PCA under dynamic
test conditions to determine the viability of the scaffold
as a potential tissue engineered blood vessel.
References.
1. Bilodeau K, Mantovani D (2006) Tissue Engineering,
12(8), 2367.
2. Zahedmanesh H et al. (In Press) Journal of Biomedical
Materials Research: Part B.
3. Campbell E et al. (2010) Proceedings of TERMIS-EU
2010 Meeting, Ireland.
Acknowledgments. Funded by Science Foundation
Ireland Research Frontiers Grant (08/RFP/ENM1378).
Keywords. Scaffolds, Cell Seeding, Mechanical
Stimulation
(18.O9) CREATING A MECHANICALLY FUNCTIONAL
DESIGN FOR PARTIAL MENISCUS REPLACEMENT
Ndreu A (1), Bahcecioglu G (1), Hasirci N (1), Hasirci V (1)
1. METU, Department of Biotechnology; Center of
Excellence on Biomaterials and Tissue Engineering,
Ankara, Turkey
Introduction. The most important meniscus property is
its ability to withstand compressive, tensile and shear
stresses. Its main functions are shock absorption, load
transmission and joint stability. When the tissue is
damaged, repair can be attempted by tissue engineering.
The aim of this study was to construct collagen-based
foams and investigate the influence of crosslinking
conditions on the properties of this potential scaffold.
Materials and Methods. Collagen type I (COLL I) was used
to prepare the foams (2 %, w/v). Both physical
(dehydrothermal, DHT) and chemical (genipin, GP, and
EDC/NHS) crosslinkings were applied. Compression (5
mm/min), tension and shear (0.5 mm/min) tests were
performed onto dry foams.
Results and Discussion. Foams were crosslinked
physically, chemically or in combination. When the
samples were crosslinked with DHT the mechanical
properties were higher than those of UXL ones. However,
DHT in combination with GP or EDC/NHS, resulted in
much higher (5 to 6-fold) compressive and tensile
properties. In the case of shear testing, crosslinkage did
not, however, significantly improve the shear properties
(Fig. 1). Meniscus regeneration is known to occur in about
6 months and the degradation rate of the material used
for engineering a tissue should be comparable with that
of the tissue formation. Even though the highest
mechanical properties were obtained with foams
crosslinked with EDC/NHS; however, a 3 month
degradation test revealed that these constructs are not
stable since almost 80 % of the foam was lost. Therefore,
double crosslinked (DHT+EDC/NHS) foams were more
appropriate. In all, compressive and shear moduli (585
kPa and 160 kPa) were above that of natural tissue (150
kPa) whereas tensile properties (2.5-4 MPa) were below
the target value (100-150 MPa). As it is, this construct is
suitable for partial meniscus replacements.
Conclusion. Foams suitable for partial meniscus
replacement were produced in this study.
Keywords. Meniscus Tissue Engineering; Mechanical
Properties
Conclusions. The ability to create customized scaffolds
with an unlimited freedom in unit cell structure can
increase the speed of research and understanding of the
influence of scaffold pore size and shape on cell
differentiation and cell growth rate.
References. 1 Hollister S.J. Scaffold Design and
Manufacturing: From Concept to Clinic. Advanced
Materials 21 (32-33), 3330-42, 2009.
Keywords. Scaffold, design, custom, personalized
A
B
C
Figure 1. Mechanical properties of COLL I-based foams.
(A) Compressive (B) tensile and (C) shear properties.
(18.O11) COMPUTER AIDED CUSTOMIZED CREATION OF
SCAFFOLDS
Verschueren P (1), Corthouts PJ (1)
1. Materialise, Belgium
Introduction. The design of scaffolds and search for the
optimal pore shape and size is a topic of ongoing research
within the tissue engineering community. In a 2009
review paper Hollister1 finds i) the need for a more
complete understanding of scaffold material and design
requirements and ii) the need to better integrate
computational design techniques with manufacturing
methods as two of the six main reasons why the
penetration of new scaffolding materials and structures
from research laboratories to the clinic has been
extremely limited.
Methods. This paper presents a method to obtain fully
customized 3D computer scaffold designs starting from
patient specific scan data. The resulting scaffolds are
ready to be produced via rapid manufacturing
techniques. This method is then illustrated on a mouse
bone scaffold coming from micro-CT scan data using
Mimics Innovation Suite software. From the virtual design
a 3D printed scaffold is created in polycaprolactone using
a fused deposition modelling technique.
Results and discussion. From patient specific data a high
quality 3D triangle mesh model is calculated. From this
model the anatomy to be replaced by a scaffold is
selected and virtually separated.
A porous unit cell which can be designed by the user, also
represented by a triangle mesh, is patterned into a
geometry which envelopes the separated anatomy from
above. A virtual cutting operation on triangle mesh level
between the separated anatomy and the patterned grid
results in a customized scaffold structure.
(18.P1) COLLAGEN-BASED SCAFFOLDS AS CARRIERS FOR
HYPOXIA-MIMICKING
BIOACTIVE
GLASSES
FOR
ORTHOPAEDIC TISSUE REGENERATION
Partap S (1), Quinlan E (1), Al Hussona M (1), Gibbons J
(1), Azevedo M (2), Stevens M (2), O'Brien FJ (1)
1. Dept of Anatomy, Royal College of Surgeons in Ireland;
2. Dept of Materials, Imperial College London, UK
A significant problem with tissue engineered constructs is
the lack of vasculature and ability to fully integrate with
the host tissue which poses one of the biggest challenges
in regenerative medicine. The Hypoxia Inducible Factor
(HIF-1a) pathway is activated under hypoxic conditions
and results in the production of pro-vasculogenic genes
such as Vascular Endothelial Growth Factor (VEGF). In our
laboratory, we have developed a series of collagen-based
scaffolds for tissue repair. The aim of this project was to
use these scaffolds as carriers for novel cobalt releasing
bioactive glasses which have shown strong potential as
hypoxia-mimicking materials by activating the HIF-1a
pathway. In this work, a bioactive glass suspension was
added to collagen and collagen-glycosaminoglycan (CG)
slurries and subsequently lyophilized. A series of variables
were examined including bioactive glass particle size (100
or 38 um diameter) and concentration (0.1, 0.2 & 0.5 mL)
as well as different constant cooling lyophilization rates
(1oC/min and 4oC/min). We found that a slower cooling
rate (1oC/min) produced a more homogenous pore
structure compared to the faster cooling rate of 4oC/min
(Fig.1). Uniaxial compression testing revealed that the
inclusion of bioactive glass significantly improved the
mechanical properties of both the collagen only and CG
scaffolds, and that the compressive moduli increased with
increasing concentration of bioactive glass added. We
also found that there was no significant effect of particle
size on the resultant properties. While porosity decreased
with increasing amounts of bioactive glass, all composites
still maintained high degrees of porosity above 97%. In
conclusion, we have successfully combined cobalt
bioactive glasses with collagen-based scaffolds. Through
ongoing research focusing on the assessment of the
cobalt bioactive glasses’ ability to induce in vitro angioand osteogenesis, we propose that these composite
scaffolds will have demonstrated potential as proangiogenic scaffolds for tissue repair.
Keywords. Collagen, Scaffold, Bioactive Glass, Hypoxia
Inducible Pathway
is important to test their mechanical properties under
confined tests. In this case, significant differences in the
aggregate modulus were detected although no changes
had been previously reported for unconfined tests.
Keywords. Scaffold, PLLA, degradation, mechanical
properties
(18.P2) EVALUATION OF MECHANICAL PROPERTIES
DURING PLLA SCAFFOLD DEGRADATION
Acosta VA (1), Mariggió D (1), Deplaine H (2), Doblaré M
(1), Gallego G (2), García-Aznar JM (3), Ochoa I (1)
1. Grupo de Mecánica Estructural y Modelado de
Materiales (GEMM) Instituto de Investigación en
Ingeniería de Aragón (I3A), Universidad de Zaragoza,
Spain; 2. Centro de Biomateriales e Ingeniería de Tejidos,
Universidad Politécnica de Valencia, Spain; 3. Grupo
MULTIESCALA EN INGENIERÍA MECÁNICA Y BIOLÓGICA
(M2BE) Instituto de Investigación en Ingeniería de Aragón
(I3A), Universidad de Zaragoza, Spain
Introduction. The application of different biodegradable
polymeric materials with three-dimensional structure to
facilitate the adhesion, diffusion and proliferation of cells
for cartilage regeneration has been widely studied (1). A
well designed scaffold should reduce, ideally, their
mechanical properties in the same rate as the tissue is
growing.
A
mechanical
and
microstructural
characterization of the scaffold degradation is important
to evaluate its future mechanobiological behavior. The
present work shows experimental parameters of PLLA
scaffold degradation (hydrolysis) under static conditions.
Materials and Methods. Scaffolds were immersed in PBS
for 6 months and preserved at 37ºC. PBS was replaced
every week. To characterize the mechanical properties of
the scaffold, uniaxial static tests like Unconfined (UC) and
Confined compression (CC) have been performed. The
Young Modulus (ES) and the Aggregate Modulus (HA) are
respectively calculated from the slope of the best linear
fit of the stress-strain graph. Poisson’s ratio (v) can be
directly deduced from ES and HA. Interconnected
porosity is an important variable in the mechanical
characterization of the scaffold. The microtomography
(Micro-CT) allows us to define the porous size, percentage
of pore structure and also to perform FE models. A
permeability test is carried out to determine how much
interconnected the porous are.
Results. A significant increase in the porous size took
place in the first time point of our study (1,5 months). A
constant increase in porous size was observed for the
whole time of the study. An exponential increase in
permeability was detected during the degradation of the
PLLA scaffold. No significant changes were observed in
the results obtained in the unconfined compression test,
as previously described in literature, but, a significant
decrease in the aggregate modulus was observed after
1,5 months.
Conclusion. Taking into account that PLLA scaffolds are
going to be located into the cartilage in a confined way, it
(18.P3) EVALUATION OF THE BIOMECHANICAL
PROPERTIES OF ARTIFICIAL SCAFFOLDS MADE OF 0.1%
FIBRIN-AGAROSE
FOR
TISSUE
ENGINEERING
APPLICATIONS
Scionti G (1), Toledano M (2), Osorio R (2), Gómez J (2),
Alaminos M (1), Campos A (1)
1. Tissue Engineering Group, Department of Histology,
University of Granada, Granada, Spain; 2. Department of
Stomatology, University of Granada, Granada, Spain
Introduction: Scaffolds made of fibrin-agarose are
characterized by high resistance, firmness and elasticity,
as fibrin is among the most resilient proteins in the
natural world; these scaffolds have shown to be
successful biomaterials in several biomedical applications,
including cornea, skin and oral-mucosa implants, because
of the great biocompatibility. The objective of this work
was to generate scaffolds made of fibrin-agarose at 0.1%
for the evaluation of their mechanical properties, to
define the potential of this biomaterial for novel
Methods: Mechanical tests were performed on the
samples: Young's modulus, stress and strain values were
determined using a tensile testing method. Due to the
lack of standardization for this kind of mechanical test on
biological materials, a new Standard Operating Procedure
for the performed experiments was developed. Different
imaging methods were used to evaluate the micro-level
network structure of the biomaterial, and the reasons
behind its biomechanical properties.
Results: The mechanical experiments showed values of
tensile stress at fracture of 0.03 MPa, Young’s modulus of
0.02 MPa and tensile strain deformation at fracture of
120%. The imaging methods showed the alignment of the
micro-fibers of the network when tensile stress is applied,
whose behavior explains the elastic properties of the
biomaterial.
Conclusions: According to these results, scaffolds made of
fibrin-agarose at 0.1% have great elasticity properties.
The mechanical behavior of this biomaterial makes it
interesting for an eventual future development of
innovative scaffolds made of fibrin-agarose, with
structure and mechanical properties with great potential
for the production of novel kinds of biomedical
applications.
Supported by grant P10-CTS-6060 from Junta de
Andalucia, Spain.
Keywords: mechanical properties, fibrin, agarose, scaffold
19. EUROSTEC: PROGRESS AND
FUTURE ASPECTS OF SOFT TISSUE
ENGINEERING FOR CHILDREN
Chair: W. Feitz
Co-chair: P. Geutjes
Keynote speaker: E. Oosterwijk
Organizer: W. Feitz
Synopsis: In this symposium new overall aspects of the
interactions and developments will be presented by the
different participating institutes (www.eurostec.eu). A
keynote lecture will be given on new scientific
developments and training methods for researchers in
the field of TERM. EuroSTEC is an Integrated Project (IP)
on ‘Soft tissue engineering for congenital birth defects in
children: from ‘biomatrix - cell interaction - model
system' to clinical trials', funded by the European
Commission under the Sixth Framework Programme
(FP6). The project brings together 15 partner
organizations (10 research institutes and 5 companies)
from 9 European countries.
Modern tissue engineering approaches are used to
develop new treatments for children with structural
disorders present at birth, such as spina bifida, urogenital
defects, gastroschisis, diaphragmatic hernia and
esophageal atresia. A translational route through in vitro
and animal experiments will lead to future clinical trials.
Ethical and regulatory issues are addressed with a
dialogue with society, including patient's associations.
Different new aspects have been studied such as microcomputed tomographical imaging of soft biological
materials using contrast techniques, human skin
substitutes, oesophagus tissue engineering, skin defects
in a fetal sheep model, and fetal mesenchymal stem cells
and new urogenital treatment options for tubular
reconstructions. Clinical trials with a main focus on fetal
intervention in case of congenital diaphragmatic hernia.
What do experts in the field think about the ethical
aspects of soft tissue engineering for congenital birth
defects in children. The EuroSTEC symposium will include
recent developments, training aspects in the field of
TERM as well as a selection of new scientific highlights in
the field of soft tissue engineering.
(19.KP) multiTERM: TRAINING MULTIDISCIPLINARY
SCIENTISTS
FOR
TISSUE
ENGINEERING
AND
REGENERATIVE MEDICINE, A MARIE CURIE INITIAL
TRAINING NETWORK. KEYNOTE PRESENTATION
Oosterwijk E (1)
1. Radboud University Medical Centre, Nijmegen,
Netherlands
Tissue engineering and regenerative medicine (TERM) is a
multidisciplinary field where scientists need to cut across
traditional fields of study. They need to understand
completely different aspects - ranging from material
choice, cell biology, to clinical translation - to successfully
design and clinically implement engineered tissue.
Unfortunately, such scientists are scarce, because such
TERM-specific interdisciplinary training is missing. To fill
the gap that currently exists, the EC has funded
MultiTERM, is a training network to provide early stage
researchers with individual and centralized training in key
elements of TERM: biomaterials, cell biology, bioreactors,
animal modeling, clinical and industrial translation.
Materials and implants for tissue engineering as well as
state-of-the-art novel visualisation procedures to monitor
the behaviour of the implanted tissues are developed by
MultiTERM participants. Here the idea behind MultiTERM
will discussed as well as results of the various studies
performed within MultiTERM.
Keywords. multidisciplinary training
(19.O1) HUMAN ECCRINE SWEAT GLAND CELLS CAN
RECONSTITUTE A STRATIFIED EPIDERMIS
Biedermann T (1), Pontiggia L (1), Böttcher-Haberzeth S
(1), Braziulis E (1), Schiestl C (1), Meuli M (1), Reichmann
E (1)
1. Tissue Biology Research Unit, Department of Surgery,
University Children's Hospital, Zurich, Switzerland
Eccrine sweat glands are generally considered to be a
possible epidermal stem cell source. Here we compared
the multilayered epithelia formed by epidermal
keratinocytes and those formed by eccrine sweat gland
cells. We demonstrated both in vitro and in vivo the
capability of human eccrine sweat gland cells to form a
stratified interfollicular epidermis substitute on collagen
hydrogels. This is substantiated by the following findings:
(1) a stratified epidermis consisting of 10-12 cell layers is
formed by sweat gland cells; (2) a distinct stratum
corneum
develops
and
is
maintained
after
transplantation onto immuno-incompetent rats; (3)
proteins such as filaggrin, loricrin, involucrin, envoplakin,
periplakin, and transglutaminases I and III match with the
pattern of the normal human skin; (4) junctional
complexes and hemidesmosomes are readily and
regularly established; (5) cell proliferation in the basal
layer reaches homeostatic levels; (6) the sweat glandderived epidermis is anchored by hemidesmosomes
within a well-developed basal lamina; and (7) palmoplantar or mucosal markers are not expressed in the
sweat gland-derived epidermis. These data suggest that
human eccrine sweat glands are an additional source of
keratinocytes that can generate a stratified epidermis.
Our findings raise the question of the extent to which the
human skin is repaired and/or permanently renewed by
eccrine sweat gland cells.
Keywords. tissue engineering, skin, human dermoepidermal skin substitute
(19.O2) DEVELOPMENT OF A NEW IN SITU PIG BLADDER
MODEL USING TISSUE ENGINEERING TECHNIQUES
Geutjes PJ (1), Janssen DAW (1), Odenthal J (1),
Heesakkers JFPA (1), Schalken JA (1), van Kuppevelt TH
(2), Feitz WFJ (1)
1. Dept. of Urology 659, Nijmegen Centre for Molecular
Life Sciences, Radboud University Nijmegen Medical
Centre, Nijmegen, The Netherlands; 2. Dept. of
Biochemistry 280, Nijmegen Centre for Molecular Life
Sciences, Radboud University Nijmegen Medical Centre,
Nijmegen, The Netherlands
Introduction. Experimental research on the urinary
bladder often requires the use of laboratory animals. For
urological research, the pig bladder is the best
translational model. However, because of practical and
financial reasons, small animal models, e.g. rats or rabbits
are often selected. With the current tissue engineering
techniques it is possible to keep tissues viable under in
vitro conditions. The aim of this study is to develop an in
situ bladder model that can be used to explore physiology
and regeneration of this organ.
Methods. Bladder mucosa was mechanically isolated
from freshly dissected abattoir pig bladders. Twenty
sterile punches (0.5 mm Ø) were taken. First, the biopsies
were cultured on 3 different substrates (i.e. type I
collagen scaffold, PET membrane and metal raster) and
cultured for 3 weeks. Secondly, five different culture
media were tested (KSFM®, SMCM®, DMEM®, RPMI®,
Epilife®). Biopsies were evaluated after different time
points (0, 2d, 1wk, 3wk, 6wk) using standard HE, scanning
electron microscopy (SEM) and immunohistochemical
staining, i.e. apoptosis (TUNEL), proliferation (Ki67) and
cell type (CK’s, αSMA, Desmin and Vimentin).
Results. Only on the type I collagen scaffolds the mucosa
remained viable for more than 3 weeks. Although smooth
muscle cells and myofibroblast were also found in the
scaffolds, the outgrowth consisted mainly out of
urothelial cells. Urothelial cells proliferated and covered
the cutting edges within 2 days. Of the 5 media used, 3
(SMC, DMEM, RPMI) were able to sustain the mucosa in
good condition with normal morphology, proliferation
(Ki67), and hardly any apoptosis (TUNEL-assay) for at least
1 week (see figure).
Conclusions. Bladder mucosa cultured on type I collagen
scaffolds under optimal circumstances, can be used as a
biological experimental model for the bladder. This new
in situ bladder model is a possible alternative for
currently used laboratory animal models.
Keywords. In situ, pig, bladder, model
(19.O3) IN VIVO IMPLANTATION OF HIGH-DENSITY
COLLAGEN GEL TUBES FOR URETHRAL REPAIR IN A
RABBIT MODEL
Micol LA (1), Arenas LF (2,3), Geutjes PJ (2,3), Hubbell JA
(1), Feitz WFJ (3), Frey P (1,4)
1. École Polytechnique Fédérale de Lausanne - EPFL,
Institute of Bioengineering, Lausanne, Switzerland;
2. Nijmegen Centre for Molecular Life Sciences - NCMLS,
Department of Biochemistry, Nijmegen, Netherlands
3. Radboud University Nijmegen Medical Centre - UMCN,
Department of Urology, Nijmegen, Netherlands; 4. Centre
Hospitalier Universitaire Vaudois - CHUV, Department of
Pediatric Urology, Lausanne, Switzerland
Urethra birth defects or injuries can impair proper
bladder voiding thus necessitating a surgical repair. Such
repair is usually undertaken using preputial skin or buccal
mucosa as donor tissue for the graft, which can lead to
long-term complications. Therefore tissue engineering is
regarded as an alternative to produce these grafts. We
had previously developed high-density collagen gel tubes
that are suitable for urinary tract tissue engineering and
can be produced, ready seeded with cells, within hours.
Our approach here was to use these constructs in vivo as
grafts for urethral repair in a rabbit model. All animals
underwent a bladder tissue biopsy by laparotomy one
month before the urethra surgery. During the urethra
surgery, high-density collagen gel tubes were implanted
in male New Zealand white rabbits after the creation of a
1cm-long urethral defect and anastomosed with fibrin
glue to the remaining urethra segments. A total of 16
animals were split into four groups, two implanted with
constructs seeded with autologous smooth muscle cells
isolated form the bladder biopsy and the two other
groups with acellular constructs. Animals were submitted
to urodymanic measurements and sacrificed 1 or 3
months after the urethra surgery depending on the
groups.
Results are expected to show a good recovery of the
urodynamics and a good tissue regeneration assessed by
histology. This in vivo study should confirm that these
high-density collagen gel tubes, which have the potential
to drastically shorten the production time of cell-seeded
tissue-engineered urinary tract grafts, are suitable for
urinary tract regeneration.
Keywords. in vivo, urethra, tissue engineering, collagen,
smooth muscle cells
(19.O4) BLADDER AUGMENTATION USING MULTIPLE
SCAFFOLDS IN ONE BLADDER AND GROWTH FACTORS IN
A PORCINE MODEL
Roelofs LAJ (1), Geutjes PJ (1), de Gier RPE (1), Farag F (1),
Tiemessen TM (1), Oosterwijk E (1), Versteeg EMM (1),
Daamen WF (1), van Kuppevelt TH (1), Kortmann BBM (1),
Feitz WFJ (1)
1. Radboud University Nijmegen Medical Centre,
Nijmegen, Netherlands
Purpose. Tissue engineering aims to develop alternatives
for the current technique of bladder augmentation. When
using a large acellular scaffold central necrosis may occur
due to the lack of cell ingrowth and blood vessel
development. In order to overcome this problem we
studied the concept of implanting multiple scaffolds in
one bladder instead of 1 large construct. Furthermore, we
studied the use of growth factors to enhance cell growth
in the scaffold.
Methods. Three different scaffolds of 3 cm Ø were
investigated: 1) crosslinked type I collagen scaffold (Col-X)
2) Col-X incorporated with heparin (Col-X-Hep) 3) Col-XHep with 3 growth factors (VEGF, FGF-2 and EGF) (Col-XHep-GF), which we compared to a ‘sham-operated’
group. In total 13 pigs were operated. Three pigs were
operated in each group and 3 scaffolds were implanted,
or 3 lesions were sutured without implant (Sham group).
Urodynamics were performed before operation. After 3
months functional (cystogram and urodynamics) and
histological evaluation (HE, CK7, vimentin, α-sma, desmin,
smoothelin) was performed on the bladders.
Results. Twelve of 13 operated pigs fulfilled the entire
experiment, one pig died because of urine leakage and
peritonitis. Survival rate was 92%. In all animals the
cystograms were normal. Urodynamic studies did not
show differences in compliance or capacity between all
groups, due to the very high compliance and capacity of
porcine bladders. Histological evaluation revealed a
normal urothelial layer and good neovascularisation in all
groups. Smooth muscle ingrowth was enhanced in the
Col-X-Hep-GF group. No signs of central maldevelopment
were seen. The scaffolds were almost fully degraded,
some remnants were visible in the Col-X-Hep group.
Conclusions. We showed the feasibility of implanting
multiple scaffolds in one bladder in order to improve its
capacity. Incorporation of heparin with growth factors
improved ingrowth of muscle cells.
Keywords. bladder augmentation collagen growth factors
(19.O5) LARGE DIAMETER TUBULAR CONSTRUCTS FOR
TISSUE ENGINEERING: SCAFFOLD PREPARATION,
CHARACTERIZATION AND CYTOCOMPATIBILITY
Hoogenkamp HR (1), Daamen WF (1), Walraven M (2),
Tiemessen TM (2), Oosterwijk E (2), van Kuppevelt TH (1),
Geutjes PJ (2), Feitz WFJ (2)
1. Department of Biochemistry 280, Nijmegen Centre for
Molecular Life Sciences, Radboud University Nijmegen
Medical Centre, 6500 HB Nijmegen, Netherlands;
2. Department of Urology 659, Nijmegen Centre for
Medical Centre, 6500 HB Nijmegen, Netherlands
Introduction. Tissue engineering can be used for
treatment of birth defects e.g. esophageal atresia or in
reconstructive surgery e.g. urinary diversion, by
developing large diameter tubular constructs which have
biological
function
and
suitable
mechanical
characteristics. In this study different tubular constructs
were developed, characterized and evaluated for
cytocompatibility.
Methods. Large tubular scaffolds (Ø 15 mm) were
prepared from highly purified bovine type I collagen with
and without commercially available synthetic polymer
mesh (Vypro-II mesh, Ethicon, Inc.), frozen in defined
moulds, lyophilized and carbodiimide-crosslinked. These
constructs were characterized by scanning electron
microscopy
(SEM),
standard
histology
(H&E),
immunofluorescent (IF) staining, TNBS assay (to assess
the degree of crosslinking), and tensile strength analysis.
Characterized scaffolds were seeded with primary porcine
epithelial cells, cultured for 1 week under static or
bioreactor conditions (Bose-ElectroForce®) and analyzed
by SEM, H&E, and IF staining.
Results. Two types of constructs were prepared with
distinct differences (Fig.1a-d). SEM and H&E showed a
highly porous network with polymer towards the outside.
Incorporation of the polymer into the collagen scaffold
significantly increased the tensile strength from 0.25±0.04
N/mm to 1.25±0.19 N/mm. Cells were evenly distributed
in the lumen (Fig.1e,f) and were positive for cytokeratin 5,
indicating epithelial phenotype.
Conclusions. In this study we successfully prepared large
tubular constructs consisting of type I collagen and
synthetic polymer mesh. The incorporation of the
polymer significantly increased the tensile strength of the
construct. Culturing under bioreactor conditions allowed
for homogeneous coverage of epithelial cells in the
lumen. From mechanical and cytocompatibility results we
conclude that large tubular collagen-polymer constructs
may be a suitable candidate scaffold for treating hollow
tubular organ defects. This study demonstrates the
feasibility of producing constructs for tubular tissue
engineering, which may lead to new approaches in
(pediatric) surgery.
Acknowledgements. This Project was financially
supported by the EuroSTEC program (LSHB-CT-2006037409).
Keywords. Tubular; Polymer; Collagen; In Vitro
(19.O6) ESOPHAGUS TISSUE ENGINEERING: IN-SITU
GENERATION OF RUDIMENTARY ESOPHAGEAL CONDUIT
USING THE FETAL MODEL
Saxena AK (1), Baumgart H (1), Tauchmann K (1),
Wiederstein I (1), Ainoedhofer H (1), Höllwarth ME (1)
1. Medical University of Graz, Austria
Background. Esophagus replacement using the present
surgical techniques is associated with significant
morbidity. Tissue engineering of the esophagus may
provide the solution for esophageal loss. In our attempts
to engineer the esophagus, this study aimed to
investigate the feasibility of generating vascularized insitu esophageal conduits using the fetal model.
Methods. Esophageal biopsies were obtained from ovine
fetus (80-120 days of gestation) and esophageal organoid
units (EOU) were proliferated. The EOU were seeded on
to bovine collagen sheets pre-seeded with fibroblasts.
After 2 weeks of maintaining the constructs in-vitro, the
constructs were tubularized on stents to create a tube
resembling the esophagus and implanted into the
omentum for in-situ tissue engineering. The edges of the
omentum were sutured using non-absorbable suture
material. The implanted constructs were retrieved after 4
weeks after birth.
Results. The omental wrap provided vascular growth
within and around the constructs as they were integrated
along the outer surface area of the scaffold. After
removal of the stents, the engineered conduit revealed a
structure similar to the esophagus. Histological
investigations demonstrated esophageal epithelium
organization into patches on the luminal side and vascular
ingrowths on the conduits’ outer perimeter.
Conclusion. Our study demonstrated the feasibility of
using the fetal ovine model for esophagus tissue
engineering. Seeding of EOU on fibroblast pre-seeded
collagen scaffolds and formation of a rudimentary conduit
resembling esophageal morphology after in-situ omental
implantation. Vascular coverage and in-growth in the
periphery of the construct could also be demonstrated.
These findings hold future promise for the engineering of
the esophagus with improved micro-architecture.
Keywords. Esophagus tissue engineering
(19.O7) EVALUATION OF LARGE TUBULAR CONSTRUCTS
FOR URINARY DIVERSION IN PIGS
Geutjes PJ (1), Roelofs LAJ (1), Hoogenkamp HH (2),
Walraven M (1), Kortmann BBM (1), de Gier RPE (1), Farag
FF (1), Tiemessen DM (1), Oosterwijk E (1), Daamen WF
(2), van Kuppevelt TH (2), Feitz WFJ (1)
1. Department of Urology 659, Nijmegen Centre for
Medical Centre, P.O Box 9101, 6500 HB Nijmegen, The
Netherlands; 2. Department of Biochemistry 280,
Nijmegen Centre for Molecular Life Sciences, Radboud
University Nijmegen Medical Centre, P.O Box 9101, 6500
HB Nijmegen, The Netherlands.
Introduction: Invasive bladder cancer usually requires
radical cystectomy followed by creation of a urostomy.
Intestinal tissue is used to create such a urinary diversion,
but complications occur in 30-80% of the patients (e.g.
infection, urinary stones, urine blockages and metabolic
disorders). Tissue engineering may be the technical
platform to develop alternatives for urological surgery.
The aim of this study is to evaluate large tubular collagenpolymer constructs (Fig. 1A) for urinary diversion in vivo.
Methods: From all female pigs (n=10), bladder biopsies
were taken and urothelial cells were isolated and
expanded. After one month, the animals received an
acellular construct (n=4), or a cell seeded construct (n=6).
To create the urostomy, the right ureter was attached to
the tubular construct with an end-to-side anastomosis.
The construct was positioned in the retroperitoneal space
(to induce blood vessel ingrowth) and fixed to the fascia
and skin (Fig. 1B). The other ureter was left intact to
enable normal voiding. After one month video
urodynamics were performed, and the animal was
sacrificed
for
further
macroscopic
and
immunohistological evaluation.
Results: Survival rate was 80% (with one related and one
unrelated death). After one month, the collagen was
resorbed and a retroperitoneal tunnel was formed which
could withstand 40 cm H2O water pressure. Although the
tunnel functioned as a urostomy, two animals had
retroperitoneal leakage and stenosis was observed in all
animals (Fig 1C). Immunohistochemistry showed
neovascularization, a moderate immune response and
formation of a neo-epithelial like layer in the lumen of the
construct. No major differences were observed between
cellular and acellular constructs.
Conclusions: The tissue engineered retroperitoneal
tunnel functioned, in most cases, as a urostomy.
Therefore, these large tubular scaffolds may be an
alternative for intestinal tissue in urostomy surgery, but
improvements are needed to reduce (skin) contractions
and fibroblast deposition and improve clinical
applicability.
Figure. Evaluation of large tubular construct for urinary
diversion: large collagen-polymer construct (A), urostomy
just after operation (B), urostomy one month after
operation (C).
(19.P1) IN VITRO CHARACTERIZATION OF HUMAN AND
PORCINE UROTHELIAL CELLS
Larsson HM (1), Gorostidi F (1,2), Barrandon Y (1,2),
Hubbell JA (1), Frey P (1,3)
1. Ecole Polytechnique Federal de Lausanne, Inst. of
Bioengineering, Lausanne, Switzerland; 2. Centre
Hospitalier Universitaire Vaudois, Dep. Anasthesiology &
Surgery, Lausanne, Switzerland; 3. Centre Hospitalier
Universitaire Vaudois, Dep. Pediatric Urology, Lausanne,
Switzerland
Introduction. An estimate of 400 million people
worldwide suffers from bladder diseases. In the case of
congenital anomalies and acquired diseases when
replacement surgery is necessary, the current treatment
plans are not optimal. Tissue-engineered constructs will
be a clinical option. The aim with tissue-engineered
constructs is that it should function throughout a
patient’s lifetime. Since, stem cells are by definition cells
able to sustain tissue homeostasis and wound healing,
they are the optimal cells to recruit or seed within the
constructs.
Method. Porcine and human urothelial cells were isolated
and cultured on irradiated 3T3-J2 fibroblasts. Growth
capacity and differentiation capacity of the cultured cells
was evaluated.
Results. We successfully cultured porcine and human
urothelial cells for 17 weeks resp. 9 weeks. Differentiation
of urothelial cells into superficial cells, as evaluated by
uroplakin-3 expression, was sparse but present in both
porcine and human cultured urothelial cells.
By clonal analysis of porcine urothelial cells, we observed
that the porcine bladder epithelium contains different
types of colony forming cells that can be further
characterized thanks to the isolation of pure clonal
populations.
Conclusion. We have started to build an argument that
we can isolate urothelial progenitor/stem cells, but a
crucial next step for the isolated cells will be to implant
the clonal cells in vivo.
Keywords. Human, Porcine, Urothelium, Progenitor/stem
cells
(19.P2) CLINICAL TESTING OF ADVANCED THERAPY
MEDICINAL PRODUCTS
Oerlemans AJM (1), Feitz WFJ (2), van Leeuwen E (1),
Dekkers WJM (1)
1. Scientific Institute for Quality of Healthcare, Radboud
University Nijmegen Medical Centre, Nijmegen, The
Netherlands; 2. Department of Urology, Radboud
University Nijmegen Medical Centre, Nijmegen, The
Netherlands
Introduction. Before December 30, 2008, tissue
engineered products fell between two legislative
categories, which prompted the creation of a new class of
medicinal products: Advanced Therapy Medicinal
Products or ATMPs, of which tissue engineered products
are a sub-category. Clinical testing is essential in the
development of any new medical technology. In
evidence-based medicine, the randomized controlled trial
(RCT) is still the gold standard. Recently, however, there
has been some debate in the literature on whether the
RCT is the best method to research treatment with
ATMPs.
A clinical application of an ATMP, currently being
researched in the EuroSTEC project, is the use of tissue
engineered constructs in children with congenital
urological defects. Ethically speaking, application of an
ATMP in this patient group creates a very specific
situation through a combination of several factors. Firstly,
we are dealing with a young child with (in the best
possible scenario) an entire life ahead of them in which
both positive and negative effects of treatment can occur.
Secondly, the characteristics of the ATMP itself: a
dynamic product with a substantial degree of variability,
that interacts with the body through an irreversible
process. Combining these features with the prerequisites
of an RCT (for example finding a suitable gold standard
treatment for the comparator arm), leads us to question
whether testing an ATMP in an RCT would be possible.
Objective. In this paper we wish to investigate whether,
given that the RCT is the gold standard in evidence-based
medicine, the RCT is also the most appropriate method to
research treatment of children with a congenital
urological defect with ATMPs. To this purpose, we will
conduct a literature study, complemented with expert
interviews.
Acknowledgments. The research for this contribution
was funded by the European Commission (EuroSTEC: EU
contract LSHB-CT-2006-037409).
Keywords. Ethics, clinical trials, ATMP
20. EXTRACELLULAR MATRIX: FROM
DEVELOPMENT BIOLOGY AT TISSUE
ENGINEERING
Chair: Sebastián San Martín
Co-chairs: Telma Zorn, Ornella Parolini
Keynote speaker: Telma Zorn
Organizer: Sebastián San Martín
Synopsis: During the morphogenesis and development of
organs, a coordinated process of proliferation and
differentiation of cells are requires. In this context,
adequate relationships with the extracellular matrix
(ECM) components are essential for embryo since the
fecundation, placentation and during the organogenesis
in mammals. The ECM comprises a variety of versatile
proteins and polysaccharides arranged in a cell surfaceassociated network. The ECM is required for many
specialised cell functions and consists of various
combinations of molecules, such as collagens,
proteoglycans and glycoproteins, which form either long
fibres or porous sheets, binding to cell surface receptors
and to other ECM components. The extracellular matrix
molecules have several function related with the
promotion of an adhesive substrate to the different types
of cells, provide structure, present growth factor to their
receptor, sequesters and stores growth factors, sense and
transducer mechanical signal. The role of the ECM and its
interaction with cells in these natural process, provide the
basic principles of material sciences that could be applied
to address mimic and explain the interaction of the cells
in artificial construct for tissue engineering.
It is well known that bioengineered tissues should
emulate the cellular and molecular structure of the native
organ, and the structure and level of differentiation of the
artificial constructs should be equivalent to those of the
tissues to be replaced. Thus, quality control of substitutes
developed by tissue engineering should verify that the
bioengineered tissues reproduce the structural patterns
of differentiation and gene expression of the native
tissue. Taking into account the importance of the ECM to
the developmental process, organization and function of
several tissues, the structure and composition of the ECM
of the bioengineered tissues developed in the laboratory
should be evaluated in vitro and in vivo models. In this
Symposium, some examples will be present that showed
as the ECM is necessary for normal development of
mammals during the pregnancy and artificial construct
develop by tissue engineering.
(20.KP) EXTRACELLULAR MATRIX REMODELLING DURING
DECIDUALIZATION IN RODENTS
Zorn TMT (1)
1. Institute of Biomedical Sciences, Universidade de São
Paulo, Brazil
Since the early stages of pregnancy the uterus is deeply
modified to acquire a favorable microenvironment to
implant and embryo by a process called decidualization.
Importantly, each uterine compartment is specifically
modified and express characteristic set of molecules,
which will play a role in the interaction between the
embryo and maternal tissues. In response to the embryo
implantation, in human and rodents, the endometrial
fibroblasts acquire an epitheliod phenotype forming the
decidual cells. Those cells originate a new and provisional
organ during pregnancy, the decidua.
Decidualization comprises cell proliferation, cell growth,
and the establishment of extensive intercellular junction
between decidual cells , that promote a deep reduction of
the extracellular spaces in the decidualized regions.
Consequently, the extracellular matrix (ECM) is under
extraordinary remodeling. The first morphological signal
of ECM remodeling in the mouse endometrium was
observed on the second day of pregnancy when collagencontaining phagossomes were seen in the cytoplasm of
the endometrial fibroblast. Moreover, the mouse decidua
is characterized by the presence of very thick collagen
fibrils with irregular profile. These thick collagen fibrils are
close related with decidual transformation since they are
exclusively found in decidualized regions. Recently, thin
serial section and double immunogold labeling
demonstrated that these thick fibrils are formed by
lateral aggregation of previously existent thin fibrils
formed at least by collagens types I, III and a
homotrimeric form of collagen type V.
Proteoglycans are also affected by decidualization. Gold
electron microscopy showed that the biglycan is
associated with thick collagen fibrils whereas decorin is
associated exclusively with thin fibrils. Finally, the estrous
cycle modulates proteoglycans expression in the mouse
uterus suggesting a role of the ovarian hormones in the
synthesis and /or degradation of these molecules.
Keywords.
Collagen,
decidua,
proteoglycans, extracellular matrix
implantation,
(20.O1) UPREGULATED EXTRACELLULAR MATRIX
COMPONENTS DURING JAW PERIOSTEAL CELL
OSTEOGENESIS
Ardjomandi N (1), Klumpp F (1), Hoffmann J (2), Reinert S
(1), Friedrich DA (1)
1. University of Tuebingen, Department of Oral- and
Maxillofacial surgery, Germany; 2. University of
Heidelberg, Department of Oral- and Maxillofacial
surgery, Germany
Objectives. The extracellular matrix and its components
have an amazing impact on cell fates like adhesion,
migration and proliferation. However, there is only a little
knowledge of the interplay of osteogenesis-related
components within the extracellular matrix. We analyzed
the expression patterns of different ECM components like
collagens and the inhibitors of metalloproteinases during
osteogenesis using jaw periosteal cells (JPC) in order to
gain more understanding of basic processes.
Methods. Gene and protein expression was analyzed at
three different time points of osteogenesis – 5, 10 and 20
days after induction and the cells were divided into 1.
untreated jaw periosteal cells, 2. osteoblast
differentiating media treated cells and 3. osteoblast
differentiating media containing BMP-2 treated cells. The
mineralization capacity of jaw periosteal cells was verified
performing alizarin red. Furthermore, the alkaline
phosphatase activity was detected.
Results. The gene expression pattern on 2D cultured OB
treated JPC that posess mineralization capacity and
enhanced alkaline phosphatase activity showed a strongly
increased induction of collagen type VII, VIII and XI in
comparison to collagen type I, where the basal levels of
untreated cells are quite high. Furthermore, it seems that
type I, VIII and XI levels are not affected by BMP-2. The
strong elevation of collagen type XI was also detected in
3D cultured cells growing within polylactic acid scaffolds.
Matrix turnover components like TIMP-4 and COMP were
also strongly upregulated during JPC osteogenesis in 2D
cultured cells, whereas in 3D culture, COMP levels were
not enhanced.
Conclusions. We were able to identify genes that are
related to the in vitro osteogenesis of jaw periosteumderived cells. These data and basic knowledge help us to
understand the process of osteogenesis in detail and to
optimize conditions for tissue engineering applications in
oral- and maxillofacial surgery using jaw periosteal cells as
a suitable stem cell source.
Keywords. Extracellular matrix components, osteogenesis
(20.O2)
MATRIX
METALLOPROTEASE-MEDIATED
CAPILLARY TUBE FORMATION IN COCULTURE OF
HUMAN BONE MARROW STROMAL CELLS AND HUMAN
UMBILICAL VEIN ENDOTHELIAL CELLS
Li H (1), Daculsi R (1), Bourget C (1), Bareille R (1), Remy M
(1), Amedee J (1)
1. INSERM 577, Bordeaux and University Victor Segalen
Bordeaux 2
Introduction. Angiogenesis is essential to tissue
reconstitution and currently represents one of the major
challenges in tissue engineering. Our previous studies
showed that the coculture of Human Bone Marrow
Stromal Cells (HBMSCs) and Human Umbilical Vein
Endothelial Cells (HUVECs) could induce capillary tube
formation, which has attracted our much interests and
the roles of different molecules in the formation of
capillary tubes have been investigated. Based on the
studies of communications between HBMSCs and
HUVECs, the current study aimed to investigate the
communication between matrix and cells, focusing on the
roles of matrix metalloproteases (MMPs) for the
formation of capillary tubes.
Methods. Cells were monocultured or cocultured in an
Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco)
supplemented with 1% (v/v) FBS. Supernatant of the
cultures were collected and cell extract were maintained
at 14 hours and 24 hours for further analysis.
Zymographic techniques, quantitative real time
polymerase chain reaction, western blot, as well as
functional studies against urokinase plasminogen
activator (uPA) were applied to measure the enzymatic
activities, expression and functionality of the MMPs.
Results. Results show that the activities of MMP-1 and
MMP-2 decreased at 24 hours and there are significant
upregulation in the enzymatic activities and expression of
MMP-2 in cocultured cells than in monocultured cells. For
MMP-1, its expression was significantly increased but its
enzymatic activities were hardly to be detected.
Expression of TIMP-1 and TIMP-2 were clearly
upregulated at 24 hours. Function studies showed that
the neutralization of uPA significantly downregulated the
expression of MMP-1 and MMP-2.
Conclusion. During the capillary tube formation in
coculture of HBMSCs and HUVECs, MMP-2 seems to play
a more important role than MMP-1. In addition, TIMP-1
and TIMP-2 acted in accordance with MMP-2. uPA has an
important effect on the regulation of MMP-2 and MMP-1.
These matrix metalloprotease activities in a coculture of
endothelial and osteoprogenitor cells could contribute to
formation a prevascular network for bone vascularized
tissue strategies.
Keywords. Matrix Metalloprotease, angiogenesis,
Cocultures, Tissue engineering
(20.O3)
DEVELOPMENT
OF
NOVEL
TYPE
FIBRINOGEN/PLDLA NANOFIBERS FOR CONTROL
ENDOTHELIAL CELLS BEHAVIOR
Gugutkov D (1), Sánchez MS (2), Altank

histology and histopathology

Transcription

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